Inhibitors of the EGF-receptor tyrosine kinase and methods for their use

ABSTRACT

Novel compounds and pharmaceutical compositions useful as EGFR tyrosine kinase inhibitors. Methods of the invention include administration of the EGFR TK inhibitors to treat diseases characterized by enhanced expression of EGF, including cancers, particularly breast cancer. Additionally, a homology model representing the structure of EGFR kinase domain is provided, which model is useful for the rationally design and screening of compounds predicted to bind favorably to EGFR and to inhibit EGFR TK.

RELATED APPLICATIONS

[0001] This application claims priority under 35 USC § 1.53(b) from U.S.patent application Ser. No. 09/273,002, filed Mar. 19, 1999, whichitself claims priority under 35 U.S.C. §119(e) from U.S. ProvisionalApplication 60/090,998, filed Jun. 29, 1998, and from U.S. ProvisionalApplication 60/097361, filed Aug. 21, 1998.

BACKGROUND OF THE INVENTION

[0002] Breast cancer is the most common form of malignancy in women,representing 32% of all new cancer cases and causing 18% of the cancerrelated deaths among women in the USA. Although the majority of patientswith metastatic breast cancer will experience an initial response,survival is only modestly improved with contemporary chemotherapyprograms. Consequently, the development of new anti-breast cancer drugshas become a high priority (Abrams, J. S., et al. M. Cancer 1994, 84,1164).

[0003] Human epidermal growth factor (EGF) is a 53 amino acid,single-chain polypeptide (Mr 6216 daltons), which exerts biologiceffects by binding to a specific cell membrane epidermal growth factorreceptor (EGFR/ErbB-1). Many types of cancer cells display enhanced EGFRexpression on their cell surface membranes (Khazaie, K., et al. R. B.Cancer & Metasis Reviews 1993, 12, 255). Enhanced expression of the EGFRon cancer cells has been associated with excessive proliferation andmetastasis (Mendelsohn, J. and Baselga, J. Biologic Therapy of Cancer:Principles & Practice 1995, 607). Examples include breast cancer,prostate cancer, lung cancer, head and neck cancer, bladder cancer,melanoma, and brain tumors (Khazaie, K., et al. R. B. Cancer & MetasisReviews 1993, 12, 255).

[0004] In breast cancer, expression of the EGFR is a significant andindependent indicator for recurrence and poor relapse-free survival(Toi, M., et al. European J. Cancer 1991, 27, 977; Chrysogelos, S. A.and Dickson, R. B. Breast Cancer Res. Treat. 1994, 29, 29; Fox, S. B.,et al. Breast Cancer Res. Treat. 1994, 29, 41). Additionally, it hasbeen shown that the EGFR has an essential function for the survival ofhuman breast cancer cells (Uckun, F. M., et al. Clin. Can. Res. 1998, 4,901; Moyer, J. D., et al. Cancer Res. 1997, 57(21), 4838). Therefore,the development of PTK inhibitors which abrogate the enzymatic functionof the EGFR tyrosine kinase has become a focal point in drug discoveryresearch programs aimed at designing more effective treatment strategiesfor metastatic breast cancer (George-Nascimento, et al. Biochemistry1988, 27, 797; Khazaie, K., et al. R. B. Cancer & Metasis Reviews 1993,12, 255; Fry, D. W. and Bridges, A. J. Curr. Opin. BiotechnoL 1995, 6,662; Wakeling, A. E., et al. Breast Cancer Research & Treatment 1996,38, 67).

[0005] The primary metabolite of the anti-inflammatory leflunomideN-(4-trifluoromethylphenyl)-5-methylisoxazol-4-carboxamide, has beenidentified as an inhibitor of the EGFR kinase (Parnham, M. J. Exp. OpinInvest. Drugs 1995, 4, 777; Xu, X., et al. Biochem. Pharmacol. 1996, 52,527; Xu, X., et al. J. Biol. Chem. 1995, 270, 12398; Bertolini, G., etal. J. Med. Chem. 1997, 40, 2011; Mattar, T., et al. A. F. E. B. S.Lett. 1993, 334, 161). Despite the identification of this inhibitor ofthe EGFR kinase, however, there is a continuing need for novelanti-cancer drugs. In particular, there is a need for anti-cancer drugswhich are more potent or more selective than existing drugs. There isalso a need for anti-cancer drugs that operate by novel mechanisms, andthus, may be useful against cancers that do not respond to, or havedeveloped resistance to, existing therapies.

SUMMARY OF THE INVENTION

[0006] Applicants have discovered compounds that selectively inhibitEGFR tyrosine kinase, without affecting the activity of other PTKs. Arepresentative compound of the invention was also found to inhibit theproliferation and in vitro invasiveness of EGFR positive human breastcancer cells at micromolar concentrations. Thus, the compounds of theinvention are useful for treating cancer (e.g. breast cancer). Thecompounds are also useful as pharmacological tools that can be used tofurther investigate EGFR kinase function, or can be used in competitivebinding assays to help identify other agents that may be useful aspharmaceuticals.

[0007] Accordingly, the invention provides a compound of the followingformula I:

[0008] where:

[0009] R₁ is (C₁-C₃)alkyl, (₃-C₆)cycloalkyl, phenyl, or NR_(a)R_(b);

[0010] R₂ is hydroxy, (C₁-C₆)alkoxy, or (C₁-C₆)alkanoyloxy;

[0011] R₃ is cyano, or (C₁-C₃)alkanoyl;

[0012] R₄ is hydrogen, or (C₁-C₃)alkyl;

[0013] R₅ is aryl, or heteroaryl;

[0014] R₁ and R_(b) are each independently hydrogen, or (C₁-C₃)alkyl; orR_(a) and R_(b) together with the nitrogen to which they are attachedare pyrrolidino, piperidino, morpholino, or thiomorpholino;

[0015] wherein any aryl, or heteroaryl of R₁ and R₅ is optionallysubstituted with one or more (e.g., 1, 2, or 3) substituentsindependently selected from halo, nitro, cyano, hydroxy,trifluoromethyl, trifluoromethyl, trifluoromethoxy, (C₁-C₃)alkoxy,(C₁-C₃)alkyl, (C₁-C₃)alkanoyl, —S(O)₂R_(c), or NR_(a)R_(b); whereinR_(c) is (C₁-C₃)alkyl, or aryl or a pharmaceutically acceptable saltthereof.

[0016] Prefereably, if R₅ is phenyl, the phenyl is substituted by—S(O)₂R_(c), or is substituted by halo and at least one othersubstituent.

[0017] Preferred compounds of formula I include those of formula II:

[0018] where R₁ is (C₁-C₆)alkyl, optionally substituted by 1, 2, or 3substituents selected from the group consisting of halo, hydroxy, amino,(C₁-C₆)alkoxy, and (C₁-C₆)alkanoyloxy; R₂ is hydroxy, (C₁-C₆)alkoxy,(C₁-C₆)alkanoyloxy;R₃ is cyano, alkanoyl; R₆ is amino, hydroxy, cyano,nitro, (C₁-C₆)alkyl, halo(C₁-C₆)alkyl, (C₁-C₆)alkoxy, halo(C₁-C₆)alkoxy,(C₁-C₆)alkanoyl, or (C₁-C₆)alkanoyloxy; and R₇ is H, NH₂, CH₃, OH, CF₃,or halo, preferably, halo is Br or Cl; or a pharmaceutically acceptablesalt thereof.

[0019] Particularly compounds of formula I include those of formulaeIII-VI:

[0020] where R₇ is H, NH₂, CH₃, OH, CF₃, or halo. Preferably, halo is Bror Cl.

[0021] where R₇ is H, NH₂, CH₃, OH, CF₃, or halo. Preferably, halo is For Cl.

[0022] where R₆ is NH—CH₃ or OCH₃.

[0023] where R₂ is —CH₂—CH₂X and X is halo, preferably F; or R₂ is—CH₂CF₃; or R₂ is:

[0024] The invention also provides a pharmaceutical compositioncomprising a compound of formula I; or a pharmaceutically acceptablesalt thereof, and a pharmaceutically acceptable carrier.

[0025] A particularly useful compound of the invention is LFM 12, havingthe structural formula:

[0026] The invention also provides a therapeutic method for treatingdiseases in which EGFR is overexpressed, particularly cancers (e.g.breast cancer) comprising administering to a mammal in need of suchtherapy, a compound of the invention, e.g., of formulae I-VI; or apharmaceutically acceptable salt thereof.

[0027] The invention also provides a compound for use in medical therapypreferably for use in treating cancer), as well as the use of a compoundof formulae I-VI for the manufacture of a medicament for the treatmentof a pathological condition or symptom in a mammal, such as a human,which is associated cancer (e.g. breast cancer).

[0028] The invention also provides a homology model representing thestructure of the EGF-R kinase domain and a docking procedure, which areuseful to rationally design compounds predicted to bind favorably toEGF-R and inhibit EGFR-TK activity. Using this model, leflunomidemetabolite analogs were designed and found to have potent inhibitoryactivity against EGFR TK (IC₅₀ value of 1.7 μM in EGF-R inhibitionassays, killing >99% of human breast cancer cells in vitro by triggeringapoptosis). New potent LFM analogs as active inhibitors of the EGF-Rtyrosine kinase are designed and confirmed using this model.

[0029] The invention also provides processes and novel intermediates,described herein, that are useful for preparing compounds of formulae I,II, IV-VI.

BRIEF DESCRIPTION OF THE FIGURES

[0030] FIGS. 1A-1C show a homology model fo the EGF-R kinase domain.FIG. 1A is a molecular surface model showing the plan as active site.FIGS. 1B and 1C are stereoviews of the catalytic site of the EGF-Rkinase domain with the inhibitor binding region represented as atriangular-shaped grid.

[0031] FIGS. 2A-2B are photographs showing the homology model of theEGFR kinase domain. FIG. 2A is a ribbon (Cα chain) representation of thehomology model of the EGFR kinase domain and a space filling model ofthe compound LFM-A12 which was docked into the catalytic site. TheN-lobe shown in blue is primarily composed of β-sheets and the C-lobeshown in gray is mostly helical. The hinge region is shown in peach.Prepared using Molscript and Raster3D programs (Bacon, D. J. andAnderson, W. F. J. Molec. Graphics 1988, 6, 219; Kraulis, P. J. Appl.Cryst. 1991, 24, 946; Merritt, E. A. and Murphy, M. E. P. Acta Cryst.1994, D50, 869).

[0032]FIG. 2B is a space filling representation of the catalytic siteresidues of the EGFR kinase domain. The Cα chain of EGFR is representedas a pink ribbon and the residues comprising the hinge region are shownin blue. One comer of the triangular binding region is located betweenthe T766 (peach) and D831 (lavender) residues. The second comerbordering the binding region is located at R817 shown in green and thethird comer is near the lower left side of the hinge region. A ball andstick model of the inhibitor LFM-A12 is shown in multicolor andrepresents a favorable orientation of this molecule in the kinase activesite of EGFR. Prepared using InsightII program (InsightII, MolecularSimulations Inc. 1996, San Diego, Calif.).

[0033]FIG. 3 shows the superimposed docked positions of the parasubstituted LFM analogs at the active site of the EGFR tyrosine kinase.The residues in the active site are shown in pink. LFM: para-CF₃substituted compound (active metabolite of leflunomide, green); LFM-A1:para-Br substituted analog (blue); LFM-A2: para-Cl substituted analog(red); LFM-A3: para-F substituted LFM analog (white). LFM-A12: para-OCF₃substituted LFM analog (gold).

[0034]FIG. 4 shows the superimposed docked positions of CF₃ substitutedLFM analogs in the active site of the EGFR tyro sine kinase. Theresidues in the active site are shown in pink. LFM: para-CF₃ substitutedcompound (active metabolite of leflunomide, green); LFM-A8: meta-CF₃substituted analog (yellow); LFM-A4: ortho-CF₃ substituted analog (red).

[0035] FIGS. 5A-5F are anti-phosphotyrosine Western blots showingselective inhibition of EGFR tyrosine kinase by LFM-A12. FIG. 5A showsEGFR immune complexes from lysates of MDA-MB-231 human breast cancercells treated with LFM or LFM-A12 for 1 hour and then assayed for tyrosine kinase activity, as described in Example 5. FIG. 5B shows a lack ofinhibition of IRK immunoprecipitated from HepG2 hepatoma cells in immunecomplex kinase assays after treatment with LFM or LFM-A12. FIG. 5C showsa lack of inhibition of HCK immunoprecipitated from transfected COS7cells in immune complex kinase assays after treatment with LFM orLFM-A12. FIGS. 5D-5F show a lack of inhibition of JAK3, JAK1, and BTKimmunoprecipitated from lysates of transfected insect ovary cells.

[0036] FIGS. 6A-6B show the docking of LFM-A12 and WHI-P154 intocatalytic sites of kinases. FIG. 6A shows the superimposed backbones ofthe catalytic site residues of the kinase domain homology models of EGFR(white), PTK (peach) and crystal structure coordinates of HCK (blue),with selected residues at positions A, B, and C. LFM-A12 is shown inmulticolor and represents its docked positionin BTK, which is alsosimilar to its docked position in HCK. The white dotted surface arearepresents the Connolly surface of LFM-A12. FIG. 6B shows superimposedbackbones of the catalytic site residues of the kinase domain homologymodels of EFGR (white), JAK3 (pink) and crystal structure coordinates ofIRK (green), with selected residues at positions A, B, and C. LFM-A12 isshown in multicolor and represents its docked positionin EGFR which isalso similar to its docked position in IRK and JAK3. The white dottedsurface area represents the Connolly surface of LFM-A12.

[0037] FIGS. 7A-7B demonstrates anti-invasive activity of LFM-A12against MDA-MB-231 human breast cancer cells. Cells were incubated withthe indicated concentrations of LFM or LFM-A12 for 24 hours,trypsinized, and placed in Boyden chambers precoated with Matrigelmatrix and allowed to migrate for 48 hours. The migrated cells werestained with Hema II solution and counted. The data points are the meanvalues from two independent experiments. Untreated breast cancer cellswere highly invasive in Matrigel-coated Boyden chambers. FIG. 7A is abar graph showing the invasion of LFM-A12-treated breast cancer cellsthrough the Matrigel matrix was inhibited in a dose-dependent fashion.The invasion of LFM-treated breast cancer cells was inhibited to alesser extent. The mean IC₅₀ values were 28.4 μM for LFM-A12 and 97.0 μMfor LFM. FIG. 7B is a series of photographs showing microscopic evidencefor the dose-dependent reduction of the numbers of migrated MDA-MB-231cells after treatment with LFM-A12.

[0038] FIGS. 8A-8B are graphs showing the cytotoxic activity of LFM andLFM-A12 against human breast cancer cells in MTT assays. MDA-MB-231 andMDA-MB-361 cells were treated with LFM (FIG. 8A) or LFM-A12 (FIG. 8B)for 36 hours in 96-well plates and the cytotoxicity was determined bythe MTT assay. The data points represent the mean (±SE) values fromthree independent experiments.

[0039] FIGS. 9A-9C are confocal images of LFM-A12—treated breast cancercells. MDA B-231 cells were treated with LFM-A3 or LFM-A12 at a finalconcentration of 100 μM for 24 hours at 37° C., as described in theExamples. After treatment, cells were processed for immunofluorescenceusing a monoclonal antibody to α-tubulin (green fluorescence). LFM-A12(FIG. 9A) but not LFM-A3 (FIG. 9C)—treated cells showed marked shrinkagewith disruption of microtubules and lost their ability to adhere to thesubstratum. Blue fluorescence represents nuclei stained with TOTO-3. Thecontrol is shown in FIG. 9A FIGS. 10A and 10B diagramatically showregions of LFM-A12 suitable for modificatin to enhance EGFR inhibition.Structural and chemical features of LFM analogs which are proposed toaid binding to the EGFR catalytic site and are described below andillustrated. Binding Mode 1, (FIG. 10A) shows the most likely mode ofbinding of the lead compound LFM-A12 at the EGFR catalytic site. Basedon the modifications of the lead compound, a second mode may also bepossible and this is illustrated in FIG. 10B (Binding Mode 2).

DETAILED DESCRIPTION OF THE INVENTION

[0040] The following definitions are used, unless otherwise described:halo is fluoro, chloro, bromo, or iodo. Alkyl, alkoxy, etc. denote bothstraight and branched groups; but reference to an individual group suchas “propyl” embraces only the straight chain group, a branched chainisomer such as “isopropyl” being specifically referred to.

[0041] It will be appreciated by those skilled in the art that compoundsof the invention having a chiral center may exist in and be isolated inoptically active and racemic forms. Some compounds may exhibitpolymorphism. It is to be understood that the present inventionencompasses any racemic, optically-active, polymorphic, orstereoisomeric form, or mixtures thereof, of a compound of theinvention, which possess the useful properties described herein, itbeing well known in the art how to prepare optically active forms (forexample, by resolution of the racemic form by recrystallizationtechniques, by synthesis from optically-active starting materials, bychiral synthesis, or by chromatographic separation using a chiralstationary phase) and how to determine a compounds ability to inhibitEGFR tyrosine kinase using the standard tests described herein, or usingother similar tests which are well known in the art.

[0042] Specific and preferred values listed below for substituents andranges, are for illustration only; they do not exclude other definedvalues or other values within defined ranges for the substituents.

[0043] The term “prodrug moiety” is a substitution group whichfacilitates use of a compound of the invention, for example byfacilitating entry of the drug into cells or administration of thecompound. The prodrug moiety may be cleaved from the compound, forexample by cleavage enzymes in vivo. Examples of prodrug moietiesinclude phosphate groups, peptide linkers, and sugars, which moietiescan be hydrolized in vivo.

[0044] Compounds of the Invention

[0045] Specific inhibitors of EGFR tyrosine kinase include those offormula I:

[0046] where:

[0047] R₁ is (C₁-C₃)alkyl, (₃-C₆)cycloalkyl, phenyl, or NR_(a)R_(b);

[0048] R₂ is hydroxy, (C₁-C₃)alkoxy, or (C₁-C₆)alkanoyloxy;

[0049] R₃ is cyano, or (C₁-C₃)alkanoyl;

[0050] R₄ is hydrogen, or (C₁-C₃)alkyl;

[0051] R₅ is aryl, or heteroaryl;

[0052] R₁ and R_(b) are each independently hydrogen, or (C₁-C₃)alkyl; orR_(a) and R_(b) together with the nitrogen to which they are attachedare pyrrolidino, piperidino, morpholino, or thiomorpholino;

[0053] wherein any aryl, or heteroaryl of R₁ and R₅ is optionallysubstituted with one or more (e.g., 1, 2, or 3) substituentsindependently selected from halo, nitro, cyano, hydroxy,trifluoromethyl, trifluoromethyl, trifluoromethoxy, (C₁-C₃)alkoxy,(C₁-C₃)alkyl, (C₁-C₃)alkanoyl, —S(O)₂R_(c), or NR_(a)R_(b); whereinR_(c) is (C₁-C₃)alkyl, or aryl or a pharmaceutically acceptable saltthereof;

[0054] Preferred are compounds of formula II, where R₁ is (C₁-C₆)alkyl,optionally substituted by 1, 2, or 3 substituents selected from thegroup consisting of halo, ΔΔΔΔ, (C₁-C₆)alkoxy, R₂ is hydroxy,(C₁-C₆)alkoxy, (C₁-C₆)alkanoyloxy; R₃ is cyano, alkanoyl; R₄ is amino,hydroxy, cyano, nitro, (C₁-C₆)alkyl, halo(C₁-C₆)alkyl, (C₁-C₆)alkoxy,halo(C₁-C₆) alkoxy, (C₁-C₆)alkanoyl, or (C₁-C₆)alkanoyloxy; and R₅ is H,NH₂, CH₃, OH, CF₃, or halo, preferably halo is Br or Cl; or apharmaceutically acceptable salt thereof.

[0055] Specifically, (C₁-C₆)alkyl can be methyl, ethyl, propyl,isopropyl, butyl, iso-butyl, sec-butyl, pentyl, 3-pentyl, or hexyl;(C₁-C₆)alkoxy can be methoxy, ethoxy, propoxy, isopropoxy, butoxy,iso-butoxy, sec-butoxy, pentoxy, 3-pentoxy, or hexyloxy; (C₁-C₆)alkanoylcan be acetyl, propanoyl or butanoyl; halo(C₁-C₆)alkyl can beiodomethyl, bromomethyl, chloromethyl, fluoromethyl, trifluoromethyl,2-chloroethyl, 2-fluoroethyl, 2,2,2-trifluoroethyl, or pentafluoroethyl;and (C₂-C₆)alkanoyloxy can be acetoxy, propanoyloxy, butanoyloxy,isobutanoyloxy, pentanoyloxy, or hexanoyloxy. A specific value for R₁ is(C₁-C₆)alkyl, optionally substituted by halo, hydroxy, amino, or(C₁-C₆)alkoxy.

[0056] Another specific value for R₁ is (C₁-C₆)alkyl.

[0057] A preferred value for R₁ is methyl.

[0058] A preferred value for R₁ is hydroxy.

[0059] A preferred value for R₃ is cyano.

[0060] A preferred value for R₄ is trifluoromethoxy.

[0061] A preferred compound is a compound of formula I wherein R₁ is(C₁-C₆)alkyl; R₂ is hydroxy, (C₁-C₆)alkoxy, or (C₁-C₆)alkanoyloxy; R₃ iscyano; and R₄ trifluoromethoxy; or a pharmaceutically acceptable saltthereof.

[0062] Another preferred compound is a compound of formula I wherein R₁is methyl; R₂ is hydroxy; R₃ is cyano; and R₄ trifluoromethoxy; or apharmaceutically acceptable salt thereof.

[0063] Preferred compounds of the invention include novel analogs of LFMdesigned to better fit the EGFR binding pocket and better interact withamino acid residues forming contacts within the pocket. These compoundsof the invention fall within four groups, or types, and are more fullydescribed below in Example 6. These compounds have the followingstructural formulae (III-VI):

[0064] where R₅ is H, NH₂, CH₃, OH, CF₃, or halo. Preferably, halo is Bror Cl.

[0065] where R₅ is H, NH₂, CH₃, OH, CF₃, or halo. Preferably, halo is For Cl.

[0066] where R₄ is NH—CH₃ or OCH₃.

[0067] where R₁ is —CH₂—CH₂X and X is halo, preferably F; or R₁ is—CH₂CF₃; or

[0068] Processes for preparing compounds of formulae I-VI are providedas further embodiments of the invention and are illustrated by thefollowing procedures and Examples in which the meanings of the genericradicals are as given above unless otherwise qualified. A compound offormula I wherein R₂ is hydroxy, can conveniently be prepared bytreating an intermediate of formula A: with an acid chloride of formula:R₁COCl.

[0069] In cases where compounds are sufficiently basic or acidic to formstable nontoxic acid or base salts, administration of the compounds assalts may be appropriate. Examples of pharmaceutically acceptable saltsare organic acid addition salts formed with acids which form aphysiological acceptable anion, for example, tosylate, methanesulfonate,acetate, citrate, malonate, tartarate, succinate, benzoate, ascorbate,ketoglutarate, and glycerophosphate. Suitable inorganic salts may alsobe formed, including hydrochloride, sulfate, nitrate, bicarbonate, andcarbonate salts.

[0070] Pharmaceutically acceptable salts may be obtained using standardprocedures well known in the art, for example by reacting a sufficientlybasic compound such as an amine with a suitable acid affording aphysiologically acceptable anion. Alkali metal (for example, sodium,potassium or lithium) or alkaline earth metal (for example calcium)salts of carboxylic acids can also be made.

[0071] The compounds of the invention may have attached theretofunctional groups to provide a prodrug derivative. The prodrugderiviative facilitates use of the drug in the body, for example, byfacilitating entry into cells. The prodrug derivative may be cleaved ornot in the active compound.

[0072] Pharmaceutical Compositions

[0073] The compounds of formula I can be formulated as pharmaceuticalcompositions and administered to a mammalian host, such as a humanpatient in a variety of forms adapted to the chosen route ofadministration, i.e., orally or parenterally, by intravenous,intramuscular, topical or subcutaneous routes.

[0074] Thus, the present compounds may be systemically administered,e.g., orally, in combination with a pharmaceutically acceptable vehiclesuch as an inert diluent or an assimilable edible carrier. They may beenclosed in hard or soft shell gelatin capsules, may be compressed intotablets, or may be incorporated directly with the food of the patient'sdiet.

[0075] For oral therapeutic administration, the active compound may becombined with one or more excipients and used in the form of ingestibletablets, buccal tablets, troches, capsules, elixirs, suspensions,syrups, wafers, and the like. Such compositions and preparations shouldcontain at least 0.1% of active compound. The percentage of thecompositions and preparations may, of course, be varied and mayconveniently be between about 2 to about 60% of the weight of a givenunit dosage form. The amount of active compound in such therapeuticallyuseful compositions is such that an effective dosage level will beobtained.

[0076] Conjugation to a Targeting Moiety

[0077] The compounds of the invention may be conjugated to a targetingmoiety for targeted delivery to a desired cell. Useful targetingmoieties are antibodies or specific ligands that bind to an antigen orligand receptor on the specific target cells. For example, the EGFR TKinhibitors of the invention may be targeted to EGFR-expressing cells byconjugation of the inhibitor to an anti-EGFR antibody, or by conjugationto EGF. Other useful targeting moieties include molecules which bind orassociate with the EGFR, including TGF-alpha, Erb2, Erb3, Erb4, orantibodies against these molecules.

[0078] To form the conjugates of the invention, a targeting moiety,which is often a polypeptide molecule, is bound to the compounds of theinvention at reactive sites, including NH₂, SH, CHO, COOH, and the like.Specific linking agents are used to link the compounds. Preferredlinking agents are chosen according to the reactive site to which thetargeting moiety is to be attached.

[0079] Methods for selecting an appropriate linking agent and reactivesite for attachment of the targeting moiety to the compound of theinvention are known, and are described, for example, in Hermanson,et.al., Bioconjugate Techniques, Academic Press, 1996; Hermanson,et.al., Immobilized Affinity Ligand Techniques, Academic Press, 1992;and Pierce Catalog and Handbook, 1996, ppT155-T201.

[0080] Pharmaceutical Additives

[0081] The tablets, troches, pills, capsules, and the like may alsocontain the following: binders such as gum tragacanth, acacia, cornstarch or gelatin; excipients such as dicalcium phosphate; adisintegrating agent such as corn starch, potato starch, alginic acidand the like; a lubricant such as magnesium stearate; and a sweeteningagent such as sucrose, fructose, lactose or aspartame or a flavoringagent such as peppermint, oil of wintergreen, or cherry flavoring may beadded. When the unit dosage form is a capsule, it may contain, inaddition to materials of the above type, a liquid carrier, such as avegetable oil or a polyethylene glycol. Various other materials may bepresent as coatings or to otherwise modify the physical form of thesolid unit dosage form. For instance, tablets, pills, or capsules may becoated with gelatin, wax, shellac or sugar and the like. A syrup orelixir may contain the active compound, sucrose or fructose as asweetening agent, methyl and propylparabens as preservatives, a dye andflavoring such as cherry or orange flavor. Of course, any material usedin preparing any unit dosage form should be pharmaceutically acceptableand substantially non-toxic in the amounts employed. In addition, theactive compound may be incorporated into sustained-release preparationsand devices.

[0082] The active compound may also be administered intravenously orintraperitoneally by infusion or injection. Solutions of the activecompound or its salts can be prepared in water, optionally mixed with anontoxic surfactant. Dispersions can also be prepared in glycerol,liquid polyethylene glycols, triacetin, and mixtures thereof and inoils. Under ordinary conditions of storage and use, these preparationscontain a preservative to prevent the growth of microorganisms.

[0083] The pharmaceutical dosage forms suitable for injection orinfusion can include sterile aqueous solutions or dispersions or sterilepowders comprising the active ingredient which are adapted for theextemporaneous preparation of sterile injectable or infusible solutionsor dispersions, optionally encapsulated in liposomes. In all cases, theultimate dosage form should be sterile, fluid and stable under theconditions of manufacture and storage. The liquid carrier or vehicle canbe a solvent or liquid dispersion medium comprising, for example, water,ethanol, a polyol (for example, glycerol, propylene glycol, liquidpolyethylene glycols, and the like), vegetable oils, nontoxic glycerylesters, and suitable mixtures thereof. The proper fluidity can bemaintained, for example, by the formation of liposomes, by themaintenance of the required particle size in the case of dispersions orby the use of surfactants.

[0084] The prevention of the action of microorganisms can be broughtabout by various antibacterial and antifungal agents, for example,parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.In many cases, it will be preferable to include isotonic agents, forexample, sugars, buffers or sodium chloride. Prolonged absorption of theinjectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminummonostearate and gelatin.

[0085] Sterile injectable solutions are prepared by incorporating theactive compound in the required amount in the appropriate solvent withvarious of the other ingredients enumerated above, as required, followedby filter sterilization. In the case of sterile powders for thepreparation of sterile injectable solutions, the preferred methods ofpreparation are vacuum drying and the freeze drying techniques, whichyield a powder of the active ingredient plus any additional desiredingredient present in the previously sterile-filtered solutions. Fortopical administration, the present compounds may be applied in pureform, i.e., when they are liquids. However, it will generally bedesireable to administer them to the skin as compositions orformulations, in combination with a detematology acceptable carrier,which may be a solid or a liquid.

[0086] Useful solid carriers include finely divided solids such as talc,clay, microcrystalline cellulose, silica, alumina and the like. Usefulliquid carriers include water, alcohols or glycols orwater-alcohol/glycol blends, in which the present compounds can bedissolved or dispersed at effective levels, optionally with the aid ofnon-toxic surfactants. Adjuvants such as fragrances and additionalantimicrobial agents can be added to optimize the properties for a givenuse. The resultant liquid compositions can be applied from absorbentpads, used to impregnate bandages and other dressings, or sprayed ontothe affected area using pump-type or aerosol sprayers.

[0087] Thickeners such as synthetic polymers, fatty acids, fatty acidsalts and esters, fatty alcohols, modified celluloses or modifiedmineral materials can also be employed with liquid carriers to formspreadable pastes, gels, ointments, soaps, and the like, for applicationdirectly to the skin of the user.

[0088] Examples of useful dermatological compositions which can be usedto deliver the compounds of formula I to the skin are known to the art;for example, see Jacquet et al. (U.S. Pat. No. 4,608,392), Geria (U.S.Pat. No. 4,992,478), Smith et al. (U.S. Pat. No. 4,559,157) and Wortzman(U.S. Pat. No. 4,820,508).

[0089] Treatment Methods

[0090] The EGFR-tyrosine kinase inhibitors of the invention are usefulto inhibit the activity of EGFR- tyrosine kinasae. In particular, thecompounds are usful in the treatment of diseases and pathologicalconditions that involve cells expressing EGFR. Most importantly, theinhibitors of the present invention provide a treatment for selectivelyinhibiting EGFR tyrosine kinase, without significant inhibition of othertyrosine kinases, such as the Src family, Tec family, and Janus familytyrosine kinases.

[0091] Many known pathologic conditions involve the EGFR and theactivity of the EGFR tyrosine kinase. These include cancers such asbreast cancer, prostate cancer, lung cancer, brain tumor, bladdercancer, and colon cancer. The EGFR TK inhibitors of the invention aredelivered to the EGFR, and particularly to the kinase binding domain ofthe receptor, in order to inhibit the kinase activity. The compounds ofthe invention may be administered to cancer patients to treat exsistingcancer, or be administered to those at risk for developing cancer,including genetic, occupational, and nutritional predisposition tocancer.

[0092] Additional disorders known to involve EGFR and its TK includeatherosclerosis, disorders of vascular smooth muscle cells, andendothelial cells involved in tumor angiogenesis.

[0093] Dosage

[0094] Useful dosages of the compounds of formula I can be determined bycomparing their in vitro activity, and in vivo activity in animalmodels. Methods for the extrapolation of effective dosages in mice, andother animals, to humans are known to the art; for example, see U.S.Pat. No. 4,938,949.

[0095] Generally, the concentration of the compound(s) of formula I in aliquid composition, such as a lotion, will be from about 0.1-25 wt-%,preferably from about 0.5-10 wt-%. The concentration in a semi-solid orsolid composition such as a gel or a powder will be about 0.1-5 wt-%,preferably about 0.5-2.5 wt-%.

[0096] The amount of the compound, or an active salt or derivativethereof, required for use in treatment will vary not only with theparticular salt selected but also with the route of administration, thenature of the condition being treated and the age and condition of thepatient and will be ultimately at the discretion of the attendantphysician or clinician. In general, however, a suitable dose will be inthe range of from about 0.5 to about 100 mg/kg, e.g., from about 10 toabout 75 mg/kg of body weight per day, such as 3 to about 50 mg perkilogram body weight of the recipient per day, preferably in the rangeof 6 to 90 mg/kg/day, most preferably in the range of 15 to 60mg/kg/day.

[0097] The compound is conveniently administered in unit dosage form;for example, containing 5 to 1000 mg, conveniently 10 to 750 mg, mostconveniently, 50 to 500 mg of active ingredient per unit dosage form.

[0098] Ideally, the active ingredient should be administered to achievepeak plasma concentrations of the active compound of from about 0.5 toabout 75 μM, preferably, about 1 to 50 μM, most preferably, about 2 toabout 30 μM. This may be achieved, for example, by the intravenousinjection of a 0.05 to 5% solution of the active ingredient, optionallyin saline, or orally administered as a bolus containing about 1-100 mgof the active ingredient.

[0099] Desirable blood levels may be maintained by continuous infusionto provide about 0.01-5.0 mg/kg/hr or by intermittent infusionscontaining about 0.4-15 mg/kg of the active ingredient(s).

[0100] The desired dose may conveniently be presented in a single doseor as divided doses administered at appropriate intervals, for example,as two, three, four or more sub-doses per day. The sub-dose itself maybe further divided, e.g., into a number of discrete loosely spacedadministrations; such as multiple inhalations from an insufflator or byapplication of a plurality of drops into the eye.

[0101] The invention may be better understood with reference to thefollowing Examples, which are not intended to limit the scope of theinvention.

Example 1 Crystal Structures of Leflunomide Metabolite and Its Analogs

[0102] Small molecule X-ray crystal structures were obtained for 10 ofthe 15 leflunomide metabolite analogs studied herein. The compounds werecrystallized using various solvents by evaporation, vapor diffusion orliquid-liquid diffusion. X-ray data on single crystals were collected ona SMART CCD detector (Bruker Analytical X-ray Systems, Madison, Wis.)using MoKα radiation (λ=0.7107 Å). The space group for each crystal wasdetermined based on systematic absences and intensity statistics. Adirect methods solution provided most of the non-hydrogen atoms from theelectron density map. Several full-matrix least squares/differenceFourier cycles were performed to locate the remaining non-hydrogenatoms. All non-hydrogen atoms were refined with anisotropic thermalparameters. Hydrogen atoms were placed in ideal positions and refined asriding atoms with relative isotropic temperature factors. The structurewas refined using full-matrix least-squares data on F².

[0103] Crystal structure calculations were performed using a SiliconGraphics INDY R4400-SC computer (Silicon Graphics Inc., Mountain View,Calif.) or Pentium computer using the SHELXTL V 5.0 (Sheldrick, G.SHELXTL Bruker Analytical X-ray Systems, Madison, Wis.) suite ofprograms. The crystal data, experimental parameters, and refinementstatistics are summarized in Table 1. TABLE 1 Crystal data, datacollection and refinement statistics for leflunomide metabolite analogs.

Compound LFM LFM-A1 LFM-A2 LFM-A7 LFM-A8 X p-CF₃ p-Br p-Cl o-F m-CF₃Empirical formula C₁₂H₉F₃N₂O₂ C₁₁H₉BrN₂O₂ C₁₁H₉ClN₂O₂ C₁₁H₉FN₂O₂C₁₂H₉F₃N₂O Crystal system Triclinic Triclinic Monoclinic MonoclinicTriclinic Space group P-1 P-1 P2₁/n P2₁/c P-1 Cell constants a =9.4862(6)Å A = 4.9906(2)Å a = 13.4297(10)Å a = 8.9641(8)Å a = 5.2176(6)Åb = 11.5511(7)Å B = 9.3735(3)Å b = 3.8073(3)Å b = 14.1215(12)Å b =10.4929(2)Å c = 12.0013(7)Å c = 11.8869(1)Å c = 21.201(2)Å c =8.3270(7)Å c = 12.0757(14)Å α = 96.126(2)° α = 77.394(2)° β = 98.779(2)°β = 101.023(2)° α = 67.719(2)° β = 105.914(1)° β = 86.4042)° β =78.921(2)° γ = 110.571(2)° γ = 88.065(2)° γ = 78.915(2)° Z 4 2 4 4 2Formula weight 270.21 281.11 236.66 220.20 270.21 Reflections 5372 27415021 5017 3660 collected Independent 3715 1836 1849 1788 1967reflections R indices R1 = 0.090 R1 = 0.062 R1 = 0.086 R1 = 0.047 R1 =0.060 (I > 2σ(I)) wR2 = 0.214 wR2 = 0.152 wR2 = 0.205 wR2 = 0.115 wR2 =0.139 Compound LFM-A9 LFM-A10 LFM-A11 LFM-A12 LFM-A13 X m-Br m-Cl m-Fp-OCF₃ 2,5-diBr Empirical formula C₁₁H₉BrN₂O₂ C₁₁H₉ClN₂O₂ C₁₁H₉FN₂O₂C₁₂H₉F₃N₂O₃ C₁₁H₈Br₂N₂O₂ Crystal system Triclinic Triclinic MonoclinicTriclinic Monoclinic Space group P-1 P-1 P2₁/c P-1 P2₁/c Cell constantsa = 5.2782(2)Å A = 5.2955(4)Å a = 4.7724(1)Å a = 4.6460(1)Å a =5.6134(1)Å b = 10.2335(4)Å B = 10.0638(7)Å b = 24.1536(1)Å b =9.0781(3)Å b = 9.9847(3)Å c = 11.5754(4)Å c = 11.2503(8)Å c = 9.1565(2)Åc = 14.6881(1)Å c = 21.5896(2)Å α = 69.792(1)° α = 103.951(2)° β =95.937(1)° α = 94.488(2)° β = 93.639(1)° β = 78.592(1)° β = 102.516(1)°β = 91.658(2)° γ = 75.837(1)° γ = 105.121(2)° γ = 93.682(2)° Z 2 2 4 2 4Formula weight 281.11 236.66 220.20 286.21 360.00 Reflections 3713 34106832 3856 5918 collected Independent 1926 1829 1851 2059 2109reflections R indices R1 = 0.056 R1 = 0.051 R1 = 0.051 R1 = 0.0741 R1 =0.040 (I > 2σ(I)) wR2 = 0.145 WR2 = 0.1277 wR2 = 0.149 wR2 = 0.18 wR2 =0.094

Example 2 Construction of a Homology Model for the EGF Receptor KinaseDomain

[0104] A homology model for the EGFR kinase domain was constructed basedon a structural alignment of the sequence of EGFR (accession # P00533,SWISS-PROT, Univ. of Geneva, Geneva, Switzerland) obtained from Genbank(National Center for Biotechnology Information, Bethesda, Md.) with thesequences of known crystal structures of other protein kinases (kinasedomains of HCK (Sicheri, F., et al. Nature 1997, 385, 602), FGFR(Mohammadi, M., et al. Science 1997, 276, 955), IRK (Hubbard, S. R. TheE. M. B. O. Journal 1997, 16, 5572), and cAPK (Zheng, J., et al. ActaCryst. 1993, D49, 362)).

[0105] A multiple sequence alignment of HCK, FGFR, IRK, and cAPK withthat of EGFR was carried out manually, conserving the overall secondarystructure within the family. Once the correspondence between amino acidsin the reference and model sequences were made, the coordinates for thestructurally conserved regions were assigned based on the coordinates ofthe reference proteins. Insertions, deletions and mutations wereincorporated into the template structure to build an initial model. Thefinal model of EGFR was subjected to energy minimization to refine themolecular structure so that any steric strain introduced during themodel-building process could be relieved (Brunger, A. T. X-PLOR 1992,New Haven, Conn.).

[0106] The model was screened for unfavorable steric contacts and, ifnecessary, such side chains were remodeled either by using a rotamerlibrary database or by manually rotating the respective side chains. Themodeling was carried out on a Silicon Graphics INDIGO2 computer (SiliconGraphics Inc., Mountain View, Calif.) using the Homology module inINSIGHTII (InsightII, Molecular Simulations Inc. 1996, San Diego,Calif.). The final homology model of the EGFR kinase domain had an rmsdeviation of 0.006 Å from ideal bond lengths and 2.0° from ideal bondangles after energy minimization. The above procedure was used toconstruct the homology model, and the homology model was used, inconjunction with small molecule crystal structures of the leflunomideanalogs, for modeling studies of the EGF-R/LFM complexes.

[0107] The modeled EGFR kinase domain has the expected protein kinasefold with the catalytic site in the center dividing the kinase domaininto two lobes. It is composed of a smaller N-terminal lobe connected bya flexible hinge (residues 764 to 773) to a larger C-terminal lobe (SeeFIGS. 1A-1B and 2A-2C). The N-terminal lobe is rich in β-strands, whilethe C-terminal lobe is mostly helical. The catalytic site is defined bytwo β-sheets that form an interface at the cleft between the two lobes.The catalytic site of the EGFR kinase domain displays a remarkablyplanar triangular binding pocket, which can bind the base ring portionof ATP. The sides of this triangle are approximately 15 Å×12 Å×12 Å andthe thickness of the binding pocket is approximately 7 Å, with anestimated volume of approximately 600 Å³. The characteristics of thistriangular region, which binds the base ring portion of ATP, waselucidated using a binding sphere surface calculated by the programSPHGEN in DOCK3.5. (Kuntz, I. D. et al., J. Mol. Biol., 1982:161:269-288). Two sides of the triangle can be visualized as beginning at anapex located between Thr⁷⁶⁶ (peach residue in FIG. 1B) and Asp⁸³¹(lavender residue in FIG. 1B), and extending towards thesolvent-accessible opening of the catalytic site. One side of thetriangle extends from the apex along the hinge region of the catalyticsite (blue residues in FIG. 1B), and a second side extends from the apexto Arg⁸¹⁷ (green residue in FIG. 1B) which is immediately adjacent tothe binding subsites for the sugar and triphosphate groups of ATP. Thehinge region of the binding site is composed of residues 764 to 773(FIG. 2). The third side of the triangle extends along the slot-shapedopening to the catalytic site.

[0108] The crystal structures of the HCK/quercetin complex (Sicheri, F.,et al. Nature 1997, 385, 602) and two FGFR/inhibitor complexes(Mohammadi, M., et al. Science 1997, 276, 955) revealed that thereported three inhibitors of HCK and FGFR bind to the catalytic sites ofthe respective tyrosine kinases. When the catalytic sites aresuperimposed with EGFR, all atoms of the three PTK inhibitors fallwithin the plane of the triangle described previously, and each moleculeis in close contact with the superimposed hinge region and Asp⁸³¹ ofEGFR. Moreover, they characteristically occupy only half of thetriangle, near the hinge region. This molecular fitting feature seems tocorrelate with tighter binding and may be an important determinant foreffective inhibitor binding. Similarly, the size and planar shape of thecatalytic site within the constructed EGFR kinase domain are likely tocontribute to its ability to form energetically favorable interactionswith planar molecules such as the LFM analogs described herein. Theseconsiderations are in agreement with conclusions derived from thestructure-activity relationship analyses of pyrolo- andpyrazoloquinazoline compounds (Palmer, B. D. et al. J. Med. Chem. 1997,40, 1519) and were therefore incorporated into the modeling strategy.

[0109] While most of the catalytic site residues of the EGFR kinasedomain were conserved relative to other PTKs, we noted a few specificvariations. EGFR residues Leu⁶⁹⁴, Val⁷⁰², Lys⁷²¹, and Ala⁷¹⁹ areconserved in EGFR, HCK, FGFR and IRK. Residues Asn⁸¹⁸ and Asp⁸³¹(opposite to the hinge) are conserved in EGFR, HCK, FGFR, IRK, BTK,JAK1, and JAK3. Residues Cys⁷⁵¹ and Thr⁸³⁰ are specific for EGFR butvary in BTK (Val, Ser), JAK1 (Val, Gly), JAK3 (Val, Ala), IRK (Val,Gly), and HCK (Val, Ala). Residues Thr⁷⁶⁶ and Leu⁷⁶⁸ in the hinge regionchanges to Met and Leu in IRK, Met and Phe in JAK1, Met and Tyr in JAK3,and to Thr and Tyr in BTK. The right side of the binding pocket (FIGS.2-4) contains Cys⁷⁷³ in EGFR and is therefore considerably morehydrophobic than the corresponding residue of PDGFR (Asp), FGFR (Asn),JAK1 (Ser), HCK (Ser), and IRK (Asp). These residue differences providea basis for designing selective inhibitors of the EGFR kinase domain.

[0110] Thus, the catalytic site of the EGFR kinase has a triangularshaped binding region which contains some specific nonconservedresidues. This information, combined with the homology model developedand described above, can be used to design compounds that will possessactivity as EGFR kinase inhibitors.

[0111] Selectivity for a particular tyrosine kinase can also be achievedby extending the binding area to the other half of the triangle(opposite the hinge), as has been observed in one of the FGF-R inhibitorstructures (Mohammadi, M., et al. Cell 1996: 86, 577-87) and (Connolly,M. L., Science, 1983, 221: 709-13). The difference in binding affinityis provided by Phe489 which extends from a beta hairpin toward theinhibitor. EGF-R has a shorter and more rigid beta hairpin that FGFRwhich would present some limitations to this strategy. The non-conservedresidue Arg 817 could also provide some binding discrimination if theinhibitor binds nearby. We concluded from our modeling studies that thecatalytic site of the EGF-R has specific features which can beadvantageous for the design of inhibitors. These features include atriangular shaped region which is accessible to an inhibitor. Wehypothesized that molecules fitting the triangular shape of the EGF-Rcatalytic site which can also form favorable contacts with the hingeregion of the binding site will bind more strongly and hence inhibitEGF-R more effectively. In order to elucidate the structure activityrelationships determining the ability of LFM to inhibit the EGF-Rtyrosine kinase as well as test the predictive value of our homologymodel of the EGF-R kinase domain, we have designed and synthesizedanalogs of this compound by systematically replacing the p-CF₃substituent in the phenyl ring. Our modeling calculations were based onthe homology model of the EGF-R kinase domain described above andcrystal structures of the LFM and its analogs. The positions of thecritical residues in the active site of the EGF-R and the dockedpositions of the LFM analogs are shown in FIGS. 2-3. The dockedpositions of the compounds indicate that the molecules maintain a closecontact with the hinge region. In all cases the nitrile nitrogen of theligand was involved in hydrogen bonding with the amide NH of Met. Inmodeling the inhibitory activity of LFM analogs with EGF-R tyrosinekinase, we calculated the binding constants (K_(i) values) based on thebinding interaction between the compounds and the catalytic site of theEGF-R kinase domain.

Example 3 Structure-Based Design and Synthesis of LFM Analogs HavingPotent EGFR-Inhibitory Activity

[0112] We hypothesized that molecules fitting the triangular shape ofthe EGFR catalytic site which can also form favorable contacts with thehinge region of the binding site would bind more strongly and henceinhibit the EGFR kinase more effectively. In modeling studies aimed atidentifying LFM analogs with a high likelihood to bind favorably to thecatalytic site of the EGFR kinase domain, we chose to calculate theK_(i) values based on the binding interaction between the inhibitor andEGFR residues. The K_(i) values were calculated for several differentinhibitors and were used to rank the predicted binding strength. Each ofthe small molecule LFM analogs described below was individually modeledinto the catalytic site of the EGFR kinase domain using an advanceddocking procedure. The position of quercetin in the HCK crystalstructure (Sicheri, F., et al. Nature 1997, 385, 602) was used as atemplate to obtain a reasonable starting point for the dockingprocedure. The various docked positions of each LFM analog wasqualitatively evaluated and consequently compared with the IC₅₀ valuesof the compounds in cell-free EGFR kinase inhibition assays. Table 2lists the interaction scores and calculated K_(i) values for LFM and itsanalogs. TABLE 2 Interaction scores, estimated K_(i) values and measuredIC₅₀ values for LFM analogs.

Cancer Cell Cytotoxicity IC₅₀ EGFR (μM) Lipo Ludi Ludi^(b) InhibitionMDA- MDA- Compound X HB^(a) Score Score K_(i)(μM) IC₅₀(μM) MB-231 MB-361LFM p-CF₃ 1 522 508 8 5.4 198.9 190.5 LFM-A1 p-Br 1 462 44932 >100 >300 >300 LFM-A2 p-Cl 1 456 443 37 >100 >300 >300 LFM-A3 p-F 1397 362 >100 >100 >300 >300 LFM-A4 o-CF₃ 1 442 354 >100 >100 >300 >300LFM-A5 o-Br 1 424 383 >100 >100 >300 >300 LFM-A6 o-Cl 1 385345 >100 >100 >300 >300 LFM-A7 o-F 1 430 416 69 74.5 >300 >300 LFM-A8m-CF₃ 1 492 465 22 >100 >300 >300 LFM-A9 m-Br 1 367354 >100 >100 >300 >300 LFM-A10 m-Cl 1 391 344 >100 >100 >300 >300LFM-A11 m-F 1 400 387 >100 >100 >300 >300 LFM-A12 p-OCF₃ 1 510 489 131.7 53.4 26.3 LFM-A13 2,5-diBr 1 436 340 >100 >100 >300 >300 LFM-A14none 1 397 367 >100 >100 >300 >300

[0113] Docking Procedure and Evaluation of Protein-InhibitorInteractions

[0114] Fixed docking in the Affinity program within InsightII(InsightII, Molecular Simulations Inc. 1996, San Diego, Calif.) was usedfor docking the LFM analogs to the EGFR tyrosine kinase catalytic site.A triangular binding region, where the base ring of ATP can bind toEGFR, was defined using a binding sphere surface calculated by theprogram SPHGEN in DOCK3.5 (Kuntz, I. D., et al. J. Mol. Biol. 1982, 161,269). The modeling calculations used to study the predicted binding ofinhibitors to EGFR were based on the homology model of EGFR describedabove and the coordinates of energy-minimized models of the compoundswhich were used for docking. The utility of the inhibitor modelcoordinates were validated by their crystal structures which wereobtained later and showed very similar molecular conformations. Each LFManalog was interactively docked into the triangular binding pocket ofEGFR based on the position of quercetin in the HCK/quercetin crystalstructure.

[0115] The hydrogens on the EGFR were generated and potentials wereassigned to both receptor and ligand prior to the start of the dockingprocedure. The docking method in the InsightII program uses the CVFFforce field and a Monte Carlo search strategy to search for and evaluatedocked structures (InsighII, Molecular Simulations Inc. 1996, San Diego,Calif.). While the coordinates for the bulk of the receptor was keptfixed, the program has the ability to define a radius of residues within5 Å distance from the LFM analog. As the modeling calculationsprogressed, the residues within the defined radius were allowed to shiftand/or rotate to energetically favorable positions to accommodate theligand. Calculations were carried out on an INDIGO2 computer (SiliconGraphics Inc., Mountain View, Calif.) using the CVFF force field in theDiscover program and Monte Carlo search strategy in Affinity (InsighII,Molecular Simulations Inc. 1996, San Diego, Calif.). No solvationprocedures were used. Conjugated gradient minimization was used toconserve CPU time, as the total number of movable atoms was greater than200. Calculations approximating hydrophobic and hydrophilic interactionswere used to determine the ten best docking positions of each LFM analogin the EGFR catalytic site. The various docked positions of each LFManalog was qualitatively evaluated using a score function in the Ludimodule (Bohm, H. J. J. Comput. Aided. Mol. Des. 1992, 6, 593; Bohm, H.J. J. Comput. Aided Mol. Des. 1994, 8, 243) of the program INSIGHTII(InsighII, Molecular Simulations Inc. 1996, San Diego, Calif.) which wasthen used to estimate a binding constant (K_(i)) for each compound inorder to rank their relative binding capabilities and predictedinhibition of EGFR. The calculated K_(i) trends for the LFM analogs werecompared with the trend of the experimentally determined tyrosine kinaseinhibition as well as cytotoxicity IC₅₀ values for the compounds, inorder to elucidate the structure-activity relationships (SAR)determining the potency of LFM analogs.

[0116] The docked inhibitors were sandwiched between two regions ofmostly hydrophobic residues. The region above the inhibitor consisted ofresidues Leu⁶⁹⁴, Val⁷⁰², Lys⁷²¹, and Ala⁷¹⁹, whereas the region belowincluded residues Leu⁸²⁰ and Thr⁸³⁰. The positions of the criticalresidues in the active site of the EGFR and the docked positions of theLFM analogs are shown in FIGS. 2-4. Of the possible orientations of theLFM analogs in this binding pocket, the one shown in FIGS. 2-4 had thehighest interaction score and was assumed by all 15 compounds. Thisindicates that the depicted docked positions represent an energeticallyfavored binding mode. In this binding mode the compounds can maintainclose contact with the hinge region on the edge of the inhibitor,residues Leu⁶⁹⁴ and Val⁷⁰² above the inhibitor, and Leu⁸²⁰ and Thr⁸³⁰below. In all cases the nitrile nitrogen of the ligand was involved inhydrogen bonding with the amide NH of Met⁷⁶⁹. In addition thepara-substituted OCF₃ group appeared to form close contacts betweenresidues Thr⁷⁶⁶ and Asp⁸³¹.

[0117] From the modeling studies, it was apparent that orthosubstitutions on the phenyl ring of LFM analogs would prevent themolecule from having close contact with the hinge region of thereceptor. The poor interaction is reflected by the higher K_(i) valuescalculated for this group of compounds (LFM-A4-A7; Table 2). The resultsindicated a trend that for a smaller substitution at the ortho position,there would be less disruption of close contacts with the receptor. Asthe substituted group got bulkier, the inhibitor would be pushed furtheraway from the hinge region of the receptor, thereby weakening thecontacts. The compound containing the smallest ortho-substituted group,LFM-A7 (with o-F) had the best interaction score in this group with acalculated K_(i) of 69 μM. Docking analysis suggested that LFM-A7 hadslightly increased contact with EGFR residues relative to theunsubstituted compound, LFM-A14, consistent with its lower calculatedK_(i) value.

[0118] The modeling also revealed that meta substituents, except form-F, would likely be sandwiched between residues Thr⁷⁶⁶ and Asp⁸³¹. Them-F group of LFM-A1 1 was predicted to be located on the opposite sideof the molecule relative to the meta-substituted groups of the other 3compounds, and docking showed that the m-F group of LFM-A11 would belocated near Asn⁸¹⁸, instead of the usual binding mode where the metagroup is located between Thr⁷⁶⁶ and Asp⁸³¹, opposite Asn⁸¹⁸. Thecalculated K_(i) values for compounds with m-F, m-Cl and m-Brsubstitutions were greater than 100 μM (Table 2). The results shown inTable 2 indicate that LFM-A8, which has a m-CF₃ substitution, is thebest fitting compound in this group with a calculated K_(i) of 22 μM.While maintaining the close contact with the hinge region, the m-CF₃substituent of LFM-A8 would extend further into a leucine-rich pocket ofthe protein (beyond Thr⁷⁶⁶ and Asp⁸³¹) and gain more hydrophobiccontact. Consequently, LFM-A8 was predicted to bind to the catalyticsite of the EGFR kinase domain better than the other meta-substitutedLFM analogs.

[0119] The modeling also revealed that the LFM analogs with parasubstitutions would have the greatest potential for inhibition of theEGFR, with p-CF₃ and p-OCF₃ being the most promising (calculatedK_(i)˜8-13 μM), followed by p-Cl and p-Br, (calculated K_(i)˜30-40 μM),and p-F (calculated K_(i)>100 μM) (Table 2). The best docked positionsof the five para substituted compounds showed a binding pattern similarto that of the meta substituted compounds except for a slight shift ofthe phenyl rings toward Thr⁷⁶⁶ (FIG. 3). The para substituted compoundswould maintain a close contact with the hinge region of the EGFR kinasedomain and be stabilized by additional contact area between the parasubstituents and the residues deep inside the binding site. The CF₃ andOCF₃ substituents in the para position would extend toward the deepestcorner of the binding site which would result in improved molecularcontact. For compound LFM-A3, the C-F bond length (1.3 Å) is shorterthan the C—Cl and C—Br bond lengths in LFM-A2 and LFM-A1 (1.8-1.9 Å) andthus the p-F group would not approach Lys⁷²¹ as closely. This weakercontact may contribute to the poor K_(i) values for LFM-A3. The parasubstituted compounds appeared to approach Asp⁸³¹ of EGFR more closelycompared to ortho or meta substituted compounds. The result of thiscloser approach may be some steric strain with Asp⁸³¹ which may forcethe residue to rotate away from the inhibitor as was observed in dockingresults. This action actually disrupts a hydrogen bond between Asp⁸³¹and Asn⁸³⁰ which could cause a slight destabilization of the proteinconformation in this region. This event is more likely to occur whenlarger para-substituted groups are involved, such as the para-Br ofLFM-A1 and para-Cl of LFM-A2. The calculations did not incorporate theenergetic effects of this speculated protein destabilization. Therefore,the true K_(i) values for compounds containing a larger para-substitutedgroup, such as LFM-A1 and LFM-A2, may be higher than the estimated K_(i)values shown in Table 2.

[0120] In these studies, the para substituted CF₃ was more active thanthe m-CF₃ compound in terms of IC₅₀ inhibition values (IC₅₀=5.4 μMvs>100 μM, Table 1), which is consistent with the modeling observationthat this compound maintains closer contacts with the hinge region thanthe m-CF₃ compound. As shown in FIG. 3, the meta substitution issterically less favorable for allowing a closer interaction with thehinge region of EGF-R relative to para substituted LFM compounds, whichmay contribute to a loss of hydrophobic contact in this region of thebinding site. This loss of hydrophobic contact would be reflected in alower calculated K_(i) value based on docking studies.

[0121] The superimposed docked positions of the three CF₃ substitutedcompounds (ortho, meta, and para) are shown in FIG. 4. The bulky orthosubstitution would prevent the ligands from having close contact withthe catalytic site and make it unlikely for this compound to showsignificant inhibitory activity. The para substituted compound, on theother hand, can maintain good contact with the hinge region of thereceptor and would likely be an effective inhibitor by our calculations.The inhibition values for meta substituted compounds were predicted tofall between those of the ortho and para substituted compounds. Thecalculated K_(i) values are consistent with their final docked positionswhich show that the m-CF₃ compound is located in-between the para andortho compounds. Docking studies also showed that the ring substituentsof most of the active compounds were predicted to be positioned betweenresidues Thr⁷⁶⁶ and Asp⁸³¹ of the EGFR.

[0122] The modeling studies suggested that LFM-A12 would exhibit potentEGFR-inhibitory activity. In order to test this hypothesis and validatethe predictive value of the described EGFR kinase domain homology model,LFM-A12, LFM, and 13 other LFM analogs listed in Table 2 were prepared.The three dimensional structures of 10 of these compounds weredetermined by single crystal X-ray diffraction. The crystal data,experimental parameters and refinement statistics are summarized inTable 1. All structures, except LFM-A2 (p-Cl), were found to beessentially planar in conformation, and all bond lengths and angles werein the expected range. For LFM-A2 the dihedral angle between thearomatic ring and side chain was close to 45°.

[0123] In all crystal structures except LFM-A 11 (m-F), the ortho ormeta substituents were found to reside on the same side of the moleculeas the nitrile group. In LFM-A11, the phenyl ring is rotated so that themeta-F and nitrile groups are on opposite sides of the molecule. Themolecular coordinates of LFM and LFM analogs which were energy-minimizedand docked into the EGFR binding site in modeling studies adopted aconformation similar to that of their crystal structures.

[0124] The effects of a compound on EGFR tyrosine kinase and survival ofhuman breast cancer cells can be determined using pharmacological modelswhich are well known to the art, or using the biological tests describedbelow.

Example 4 Chemistry and Synthesis

[0125] All chemicals were purchased from Aldrich (Milwaukee, Wis.) andwere used without further purification. Except where noted, eachreaction vessel was secured with a rubber septum, and the reaction wasperformed under nitrogen atmosphere. ¹H spectra were obtained on aVarian Mercury 300 instrument spectrometer (Palo Alto, Calif.) atambient temperature in the solvent specified. Melting points weredetermined using a Fisher-Johns melting point apparatus and areuncorrected. FT-IR spectra were recorded on a Nicolet Protege 460spectrometer (Madison, Wis.). GC/MS spectra were obtained on a HP 6890GC System (Palo Alto, Calif.) equipped with a HP 5973 Mass SelectiveDetector.

[0126] Scheme 1 shows the general synthetic scheme for the preparationsof LFM, and LFM-A1-LFM-A14 (Kuo, E. A., et al. J. Med. Chem. 1996, 39,4608; Sjogren, E. R., et al. J. Med. Chem. 1991, 34, 3295). Cyanoaceticacid 1 was coupled with the REQUISITE substituted-aniline 2 in thepresence of diisopropylcarbodiimide (DIC) to form 3. Compound 3 wastreated with NaH and acylated with acetyl chloride to afford the finalproducts.

[0127] General Synthetic Procedures

[0128] The general synthetic procedures are described in the literature.(Kuo, E. A., et al. J. Med. Chem. 1996, 39, 4608; Sjogren, E. R., et al.J. Med. Chem. 1991, 34, 3295). In general, 1,3-diisopropylcarbodiimide(1.75 g; 13.9 mmol) was added to a solution of cyanoacetic acid 1 (1.70g; 20.0 mmol) and the requisite substituted-aniline 2 (12.6 mmol) intetrahydrofuran (25 mL) at 0° C. The mixture was stirred for 12 hours atroom temperature.

[0129] The urea precipitate (reaction side product) was removed byfiltration and the filtrate was partitioned between ethyl acetate and0.5 N HCl. The organic layer was sequentially washed with brine twice,dried over anhydrous Na₂SO₄ and concentrated by rotary-evaporation. Thesolid product was recrystallized from ethyl alcohol to give pure 3.Sodium hydride (0.93 g; 60% in mineral oil; 23.2 mmol) was added slowlyto the solution of 3 (12.0 m mol) in tetrahydrofuran (40 mL) at 0° C.After stirring for 30 minutes at 0° C., the requsite acid chlorideR₁COCl (1.04 g; 13.2 mmol) was added to the reaction mixture. Thereaction was continued for another hour at room temperature and then wasquenched by the addition of acetic acid (2 mL). The mixture was pouredinto ice water (100 mL) containing 2.5 mL of hydrochloric acid toprecipitate the crude product, which was collected by filtration andwashed with water. The final pure product was obtained byrecrystallization.

[0130] Physical Data of Specific Compounds:

[0131]α-Cyano-β-hydroxy-β-methyl-N-[4-(trifluoromethyl)phenyl]-propenamide(LFM). mp: 230-233° C.; IR (KBr): 3303, 2218, 1600 and 1555 cm⁻¹; ¹H NMR(DMSO-d₆): δ 11.01 (s, 1H, NH), 7.75 (d,J=8.4 Hz, 2H, ArH), 7.64 (d,J=8.4 Hz, 2H, ArH), 2.22 (s, 3H, CH₃); GC/MS m/z 270 (M⁺), 161, 142,111.

[0132] α-Cyano-β-hydroxy-β-methyl-N-(4-bromophenyl)propenamide (LFM-A1).mp: 213-214° C.; IR (KBr): 3288, 2228, 1615, 1555 cm⁻¹; ¹H NMR(DMSO-d₆): δ 10.51 (s, 1H, NH), 7.49 (s, 4H, ArH), 2.25 (s, 3H, CH₃); MS(EI) m/z 282(M⁺+z), 280 (M⁺), 173, 171.

[0133] α-Cyano-β-hydroxy-β-methyl-N-(4-chlorophenyl)propenamide(LFM-A2). mp: 209-211° C.; IR(KBr): 3298, 2223, 1598 and 1552 cm⁻¹; ¹HNMR (DMSO-d₆): δ 10.48 (s, 1H, NH), 7.54 (d, J=8.7 Hz, 2H, ArH), 7.45 (sbr, 1H, OH), 7.36 (d, J=8.7 Hz, 2H, ArH), 2.25 (s, 3H, CH₃); MS (CI) m/z236 (M⁺), 121, 127.

[0134] α-Cyano-β-hydroxy-β-methyl-N-(4-fluorophenyl)propenamide(LFM-A3). mp: 165-166° C.; IR (KBr): 3298, 2218, 1610 and 1560 cm⁻¹; ¹HNMR (DMSO-d₆): δ 10.33 (s, 1H, NH), 7.80 (s br, 1H, OH), 7.53 (m, 2H,ArH), 7.16 (m, 2H, ArH), 2.26 (s, 3H, CH₃); GC/MS m/z 220 (M⁺), 111.

[0135]α-Cyano-β-hydroxy-β-methyl-N-[2-(trifluoromethyl)phenyl]-propenamide(LFM-A4). mp: 61-63° C.; IR (KBr): 3435, 2209, 1619, 1952 and 1548 cm⁻¹;¹H NMR (DMSO-d₆): δ 10.99 (s, 1H, NH), 8.03 (d, J=7.5 Hz, 1H, ArH), 7.67(d, J=7.5 Hz, 1H ArH), 7.60 (dd, J=7.5, 7.5 Hz, 1H, ArH), 7.29 (dd,J=7.5, 7.5 Hz, 1H, ArH) 5.71 (s br, 1H OH), 2.20 (s, 3H, CH₃); GC/MS m/z270 (M⁺), 161, 141, 114.

[0136] α-Cyano-β-hydroxy-β-methyl-N-(2-bromophenyl)propenamide (LFM-A5).mp: 98-100° C.; IR (KBr): 3351, 2214, 1609, 1585 and 1536 cm⁻¹; ¹H NMR(DMSO-d₆): δ 10.76 (s, 1H, NH), 8.06 (dd, J=8.1, 1.5 Hz, 1H, ArH), 7.62(dd, J=8.1, 1.5 Hz, 1H, ArH), 7.33 (m, 1H, ArH), 7.03 (m, 1H, ArH), 6.60(s br, 1H, OH), 2.22 (s, 3H, CH₃); ); MS (EI) m/z 282(M⁺+z), 280 (M⁺),173, 171.

[0137] α-Cyano-β-hydroxy-β-methyl-N-(2-chlorophenyl)propenamide(LFM-A6). mp: 93-94° C.; IR (KBr): 3372, 2208, 1644, 1621 and 1587 cm⁻¹;¹H NMR (DMSO-d₆): δ 10.96 (s, 1H, NH), 8.16 (d, J=8.1 Hz, 1H, ArH), 7.46(dd, J=7.5, 1.5 Hz, 1H, ArH), 7.29 (m, 1H, ArH), 7.08 (m, 1H, ArH), 2.22(s, 3H, CH₃); MS (CI) m/z 236 (M⁺), 29, 127.

[0138] α-Cyano-β-hydroxy-β-methyl-N-(2-fluorophenyl)propenamide(LFM-A7). mp: 118-119° C.; IR (KBr): 3409, 2212, 1613, 1591 and 1532cm⁻¹; ¹H NMR (DMSO-d₆): δ 10.70 (s, 1H, NH), 7.91 (m, 1H, ArH), 7.23 (M,IH, ArH), 7.13 (m, 2H, ArH), 7.10 (s br, 1H, OH), 2.22 (s, 3H, CH₃);GL/MS m/z 220 (M⁺), 111.

[0139]α-Cyano-β-hydroxy-β-methyl-N-[3-(trifluoromethyl)phenyl]-propenamide(LFM-A8). mp: 182-184° C.; IR (KBr): 3303, 2221, 1619 and 1572 cm⁻¹; ¹HNMR (DMSO-d₆): δ 10.79 (s, 1H, NH), 8.05 (s br, 1H, OH) 8.04 (s, 1H,ArH), 7.75 (d, J=8.1 Hz, 1H, ArH), 7.53 (dd, J=8.1, 7.5 Hz, 1H, ArH),7.42 (d, J=7.5 Hz, 1H, ArH), 2.24 (s, 3H, CH₃); GL/MS m/z 270 (M⁺), 161.

[0140] α-Cyano-β-hydroxy-β-methyl-N-(3-bromophenyl)propenamide (LFM-A9).mp: 184-185° C.; IR (KBr): 3303, 2228, 1610, 1595 and 1550 cm⁻¹; ¹H NMR(DMSO-d₆): δ 10.56 (s, 1H, NH), 7.89 (m, 1H, ArH), 7.47 (m, 1H, ArH),7.28 (m, 2H, ArH), 6.37 (s br, 1H, OH), 2.26 (s, 3H, CH₃); MS (EI) m/z282 (M⁺+H, ⁸¹Br), 280 (M⁺+H, ⁷⁹Br), 171, 173.

[0141] α-Cyano-β-hydroxy-β-methyl-N-(3-chlorophenyl)propenamide(LFM-A10). mp: 184-187° C.; IR (KBr): 3293, 2221, 1610, 1595 and 1557cm⁻¹; ¹H NMR (DMSO-d₆): δ 10.61 (s, 1H, NH), 7.76 (m, 1H, ArH), 7.42 (m,1H, ArH), 7.33 (M, 1H, ArH), 7.16 (m, 1H, ArH), 6.84 (S br, 1H, OH),2.25 (s, 3H, CH₃); MS (CI) m/z 236 (M⁺).

[0142] α-Cyano-β-hydroxy-β-methyl-N-(3-fluorophenyl)propenamide(LFM-A11). mp: 136-138° C.; IR (KBr): 3297, 2221, 1613, 1597 and 1567cm⁻¹; ¹H NMR (DMSO-d₆): δ 10.54 (s, 1H, NH), 7.54 (m, 1H, ArH), 7.33 (m,2H, ArH), 6.93 (m, 1H, ArH), 2.27 (s, 3H, CH₃); GL/MS m/z 220 (M⁺), 111.

[0143]α-Cyano-β-hydroxy-β-methyl-N-[4-(trifluoromethoxy)phenyl]-propenamide(LFM-A12). mp: 182-183° C.; IR (KBr): 3308, 2213, 1625 and 1580 cm⁻¹; ¹HNMR (DMSO-d₆): δ 10.57 (s, 1H, NH), 7.90 (s br, 1H, OH), 7.64 (d, J=8.7Hz, 2H, ArH), 7.32 (d, J=8.7 Hz, 2H, ArH), 2.25 (s, 3H, CH₃); GL/MS m/z286 (M⁺), 177, 108.

[0144] α-Cyano-β-hydroxy-β-methyl-N-(2,5-dibromophenyl)propenamide(LFM-A13). mp: 148-150° C.; IR (KBr): 3353, 2211, 1648 and 1590 cm⁻¹; ¹HNMR (DMSO-d₆): δ 11.41 (s, 1H, NH), 8.57 (d, J=2.4 Hz, 1H, ArH), 7.55(d, J=8.7 Hz, 1H, ArH), 7.14 (dd, J=8.7, 2.4 Hz, 1H, ArH), 7.10 (s br,1H, OH), 2.17 (s, 3H, CH₃); MS (EI) m/z 362 (M⁺+4), 360 (M⁺+2), 358(M⁺), 253, 251, 249, 150.

[0145] α-Cyano-β-hydroxy-β-methyl-N-(phenyl)propenamide (LFM-A14). mp:134-135° C.; IR (KBr): 3281, 2214, 1605, 1579 and 1554 cm⁻¹; ¹H NMR(DMSO-d₆): δ 10.33 (s, 1H, NH), 7.51 (d, J=7.5 Hz, 2H, ArH), 7.40 (s br,1H, OH), 7.31 (dd, J=7.5, 7.5 Hz, 2H, ArH), 7.11 (m, 1H, ArH), 2.26 (s,3H, CH₃); GL/MS m/z 202 (M⁺), 93.

Example 5 Biological Tests

[0146] Immunoprecipitation of Recombinant Proteins from Insect Cells

[0147] Sf21 cells were infected with a baculovirus expression vector forBTK, JAK1, or JAK3, as previously reported (Vassilev, A., et al. J.Biol. Chem. 1998, 274, 1646-1656; Goodman, P. A., et al. J. Biol. Chem.1998, 273, 17742). Cells were harvested and lysed (10 mM Tris pH 7.6,100 mM NaCl, 1% Nonidet P-40, 10% glycerol, 50 mM NaF, 100 mM Na₃VO₄, 50mg/ml phenylmethylsulfonyl fluoride, 10 mg/ml aprotonin, 10 mg/mlleupeptin) and the kinases were immunoprecipitated from the lysates, asreported (Vassilev, A., et al. J. Biol. Chem. 1998, 274, 1646-1656).

[0148] Antibodies used for immunoprecipitations from insect cells are asfollows: Polyclonal rabbit anti-BTK serum (Mahajan, S., et al. Mol.Cell. Biol. 1995, 15, 5304), polyclonal rabbit anti-JAK1 (HR-785), cat#sc-277, rabbit polyclonal IgG affinity purified, 0.1 mg/ml, Santa CruzBiotechnology; and polyclonal rabbit anti-JAK3 (C-21, cat # sc-513,rabbit polyclonal IgG affinity purified, 0.2 mg/ml, Santa CruzBiotechnology). Kinase assays were performed following a 1 hour exposureof the immunoprecipitated tyrosine kinases to the test compounds, asdescribed in detail elsewhere (Mahajan, S., et al. Mol. Cell. Biol.1995, 15, 5304; Uckun, F. M., et al. Science 1996, 22, 1096). Theimmunoprecipitates were subjected to Western blot analysis as previouslydescribed (Vassilev, A., et al. J. Biol. Chem. 1998, in press).

[0149] Cell Lines, Reagents, and Biochemical Assays

[0150] MDA-MB-231 (ATCC HTB-26) and MDA-MB-361 (ATCC HTB-27) are EGFRpositive human breast cancer cell lines (Uckun, F. M., et al. Clin. Can.Res. 1998, 4, 901). These cell lines were maintained in RPMI 1640 mediumsupplemented with 10% fetal bovine serum. For subculturing, medium wasremoved from the flasks containing a confluent layer of cells, and fresh0.25% trypsin was added for 1-2 min. Trypsin was removed and culturesincubated for 5-10 min. at 37° C. until cells detached. Fresh medium wasthen added, and cells were aspirated and dispensed into new flasks.COS-7 simian kidney cell line and HepG2 human hepatoma cell line wereobtained from ATCC.

[0151] Antibodies directed against BTK, JAK1, JAK3, and HCK have beendescribed previously (Vassilev, A., et al. J. Biol. Chem. 1998, inpress; Goodman, P. A., et al. J. Biol. Chem. 1998, 273, 17742; Mahajan,S., et al. Mol. Cell. Biol. 1995, 15, 5304; Uckun, F. M., et al. Science1996, 22, 1096). Polyclonal antibodies to BTK were generated byimmunization of rabbits with glutathione S-transferase (GST) fusionproteins (Pharmacia Biotech Inc.) containing the first 150 amino acidsof BTK. The monoclonal anti-Fas antibody (F22120) was obtained from theTransduction Laboratories, Inc. (Lexington, Ky.).

[0152] Immunoprecipitations, immune-complex protein kinase assays, andimmunoblotting using the ECL chemiluminescence detection system(Amersham Life Sciences) were conducted as described previously(Vassilev, A., et al. J. Biol. Chem. 1998, in press; Goodman, P. A., etal. J. Biol. Chem. 1998, 273, 17742; Mahajan, S., et al. Mol. Cell.Biol. 1995, 15, 5304; Uckun, F. M., et al. Science 1996, 22, 1096).

[0153] Horse radish peroxidase-conjugated sheep anti-mouse, donkeyanti-rabbit secondary antibodies and ECL reagents were purchased fromAmersham (Oakbrook, Ill.). For insulin receptor kinase (IRK) assays,HepG2 human hepatoma cells grown to approximately 80% confluency werewashed once with serum-free DMEM and starved for 3 hour at 37° in a CO₂incubator. Subsequently, cells were stimulated with insulin (Eli Lilly,cat# CP-410;10 units/ml/10×10⁶ cells) for 10 minutes at roomtemperature. Following this IRK activation step, cells were washed oncewith serum free medium, lysed in NP-40 buffer and IRK wasimmunoprecipitated from the lysates with an anti-IRb antibody (SantaCruz, Cat.# sc-711, polyclonal IgG). Prior to performing theimmunecomplex kinase assays, the beads were equilibrated with the kinasebuffer (30 mM Hepes pH 7.4, 30 mM NaCl, 8 mM MgCl2, 4 mM MnCl₂).

[0154] For HCK kinase assays, we used HCK-transfected COS-7 cells. Thecloning and expression of HCK in COS-7 cells has been describedpreviously (Saouaf, S. J., et al. J. Biol. Chem. 1995, 270, 27072). ThepSV7c-HCK plasmid was transfected into 2×10⁶ COS-7 cells usingLipofectamine (GIBCO/BRL), and the cells were harvested 48 hours later.The cells were lysed in NP-40 buffer and HCK was immunoprecipitated fromthe whole cell lysates with an anti-HCK antibody.

[0155] Immune-Complex Kinase Assays and Anti-PhosphotyrosineImmunoblotting

[0156] For EGFR immune complex kinase assays, 24-hours after treatmentwith leflunomide analogs, MDA-MB-231 breast cancer cells were stimulatedwith 20 ng/mL EGF for 5 minutes, lysed in 1% Nonidet-P-40 buffer, andcell lysates were immunoprecipitated with an anti-EGFR antibody reactivewith the sequence Ala³⁵¹-Asp³⁶⁴ of the human EGFR (Upstate BiotechnologyInc. [UBI] Catalog # 05-104) (Uckun, F. M., et al. Clin. Can. Res. 1998,4, 901). EGFR immune complexes were examined for tyrosinephosphorylation by Western blot analysis, as described by Uckun, F. M.,et al. Clin. Can. Res. 1998, 4, 901. All anti-phosphotyrosine Westernblots were subjected to densitometric scanning using the automated AMBISsystem (Automated Microbiology System, Inc., San Diego, Calif.) and foreach time point a % inhibition value was determined by comparing thedensity ratios of the tyrosine phosphorylated EGFR protein bands tothose of the baseline sample and using the formula: %Inhibition=100−100×[Density of tyrosine phosphorylated EGFRband]_(test sample)/[Density of tyrosine phosphorylated EGFRband]_(baseline control sample). The IC₅₀ values were determined usingan Inplot program (Graphpad Software, Inc., San Diego, Calif.).

[0157] In other experiments, MDA-MB-231 cells were stimulated with 10ng/ml EGF prior to immunoprecipitation of the EGFR. EGFR immunecomplexes were incubated for 1 hour at room temperature with various LFManalogs and tyrosine kinase assays were performed in the presence of[γ-³²P]-ATP, as previously described (Uckun, F. M., et al. Clin. Can.Res. 1998, 4, 901; Narla, R. K., et al. Clin. Can. Res 1998, 1405;Uckun, F. M., et al. Science 1996, 22, 1096; Uckun, F. M., et al.Science 1995, 267, 886). The kinase assay gels were analyzed both byautoradiography and using the BioRad Storage Phosphor Imager System(BioRad, Hercules, Calif.) for quantitative scanning.

[0158] In Vitro Treatment of Cells with LFM Compounds

[0159] In order to determine the cytotoxic activity of LFM and itsanalogs against breast cancer cells, cells in alpha-MEM supplementedwith 10% (v/v) fetal calf serum were treated with various concentrationsof the compounds for 24 hours at 37° C., washed twice in alpha-MEM, andthen used in either MTT assays or in vitro invasion assays, as describedhereinafter.

[0160] Cytotoxicity Assay

[0161] The cytotoxicity of various compounds against human breast cancercell lines was analyzed using the MTT(3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assay(Boehringer Mannheim Corp., Indianapolis, Ind.). Uckun F. M., et al.Clin. Cancer Res. 1998, 4, 901-912. Exponentially growing breast cancercells were seeded into a 96-well plate at a density of 2.5×10⁴cells/well and incubated for 36 hours at 37° C. prior to drug exposure.On the day of treatment, culture medium was carefully aspirated from thewells and replaced with fresh medium containing the LFM analogs atconcentrations ranging from 2 to 250 μM. Triplicate wells were used foreach treatment. The cells were incubated with the various compounds for36 hours at 37° C. in a humidified 5% CO₂ atmosphere. To each well, 10μl of MTT (0.5 mg/ml final concentration) was added and the plates wereincubated at 37° C. for 4 hours to allow MTT to form formazan crystalsby reacting with metabolically active cells. The formazan crystals weresolubilized overnight at 37° C. in a solution containing 10% SDS in 0.01M HCl. The absorbence of each well was measured in a microplate reader(Labsystems) at 540 nm and a reference wavelength of 690 nm.

[0162] To translate the absorbance A₅₄₀ values into the number of livecells in each well, the A₅₄₀ values were compared to those on standardA₅₄₀- versus - cell number curves generated for each cell line. Thepercent survival was calculated using the formula: % survival=Live cellnumber[test]/Live cell number [control]×100. The IC₅₀ values werecalculated by non-linear regression analysis using an Graphpad Prismsoftware version 2.0 (Graphpad Software, Inc., San Diego, Calif.).

[0163] Confocal Laser Scanning Microscopy

[0164] Immunofluorescence was used to examine the morphologic featuresof breast cancer cells treated with LFM and its analogs. Before theexperiment, cells were trypsinized from rapidly growing tissue cultureflasks and seeded onto sterile 22 mm² coverslips in 6-well cultureplates. Cells on coverslips were returned to the incubator for 24 hoursprior to treatment. The following day, drugs were added from a stocksolution made in DMSO to a final concentration of 100 μM. Final DMSOconcentration was 0.1% in both test samples and controls. Cells werereturned to a 37° C. incubator for 24 hours before further processing.

[0165] At 24 hours, coverslips were fixed in −20° C. methanol for 15minutes followed by a 15 minute incubation in phosphate buffered saline+0.1% Triton X-100(PBS-Tx). Coverslips were next incubated with amonoclonal antibody against α-tubulin (Sigma Chemical Co, St. Louis,Mo.) at a dilution of 1:1000 for 40 minutes in a humidified chamber at37° C. Coverslips were washed for 15 minutes in PBS-Tx followed by a 40min incubation with a goat anti-mouse IgG antibody conjugated to FITC(Amersham Corp., Arlington Heights, Ill.). The coverslips were againrinsed in PBS-Tx and incubated with 5 μM TOTO-3 (Molecular Probes,Eugene Oreg.) for 20 minutes to label the nuclear DNA. Coverslips wereimmediately inverted onto slides in Vectashield (Vector Labs,Burlingame) to prevent photobleaching, sealed with nail varnish andstored at 4° C. Slides were examined using a Bio-Rad MRC-1024 LaserScanning Confocal Microscope mounted on a Nikon Eclipse E800 uprightmicroscope with high numerical aperture objectives. Digital data wasprocessed using Lasersharp (Bio-Rad, Hercules Calif.) and AdobePhotoshop softwares (Adobe Systems, Mountain View, Calif.) and printedon a Fuji Pictography thermal transfer printer (Fuji, Elmsford, N.Y.).

[0166] Apoptosis Assays

[0167] Loose packing of membrane phospholipid head groups and cellshrinkage precede DNA fragmentation in apoptotic cells, therebyproviding MC540 binding as an early marker for apoptosis (Uckun F. M.,et al. Clin. Cancer Res. 1998, 4, 901-912; and Kuo, E. A. J. Med. Chem.1996, 39, 4608-4621). Plasma membrane permeability to propidium iodide(PI, Sigma) develops at a later stage of apoptosis (Uckun F. M., et al.Clin. Cancer Res. 1998, 4, 901-912; and Kuo, E. A. J. Med. Chem. 1996,39, 4608-4621). MC540 binding and PI permeability were simultaneouslymeasured in breast cancer cells 24 hours after exposure to leflunomideanalogs, as described (Uckun F. M., et al. Clin. Cancer Res. 1998, 4,901-912. Stock solutions of MC540 and PI, each at 1 mg/mL, were passedthrough a 0.22 μm filter and stored at 4° C. in the dark. Shortly beforeanalysis, suspensions containing 1×10⁶ cells were suspended in 5 μg/mLMC540 and 10 μg/mL PI and kept in the dark at 4° C. Whole cells wereanalyzed with a FACS Calibur or FACS Vantage flow cytometer (BectonDickinson, San Jose, Calif.). All analyses were done using 488 nmexcitation from an argon laser. MC540 and PI emissions were split with a600 nm short pass dichroic mirror and a 575 nm band pass filter wasplaced in front of one photomultiplier tube to measure MC540 emissionand a 635 nm band pass filter was used for PI emission.

[0168] Clonogenic Assays

[0169] After treatment with LFM analogs, cells were resuspended inclonogenic medium consisting of alpha-MEM supplemented with 0.9%methylcellulose, 30% fetal bovine serum, and 50 μM 2-mercaptoethanol.Cells were plated in duplicate Petri dishes at 100,000 cells/mL/dish andcultured in a humidified 5% CO2 incubator for 7 days. Cancer cellcolonies were enumerated on a grid using an inverted phase microscope ofhigh optical resolution (Uckun F. M., et al. Clin. Cancer Res. 1998, 4,901-912; and Kuo). Results were expressed as % inhibition of clonogeniccells at a particular concentration of the test agent using the formula:% Inhibition=(1-Mean # of colonies [Test]/Mean # of colonies[Control])×100. Furthermore, the dose survival curves were constructedusing the percent control survival (=Mean # of colonies[Test]/Mean # ofcolonies [Control]×100) results for each drug concentration as the datapoints and the IC50 values were calculated. The IC50 values weredetermined using an Prism Version II Inplot program (Graphpad Software,Inc., San Diego, Calif.).

[0170] Transfilter Cell Invasion Assays

[0171] The in vitro invasiveness of MDA-MB-231 human breast cancer cellswas assayed using a previously published method, which employsMatrigel-coated Costar 24-well transwell cell culture chambers (“Boydenchambers”) with 8.0 μm pore polycarbonate filter inserts which have beendemonstrated to permit the migration of human cancer cells (Yoshida, D.,et al. Neurosurgery 1996, 39, 360). The chamber filters were coated with50 μg/ml of Matrigel matrix, incubated overnight at room temperatureunder a laminar flow hood and stored at 4° C.

[0172] On the day of the experiment, the coated inserts were rehydratedwith 0.5 ml serum-free DMEM containing 0.1% bovine serum albumin for 1-2hours. To study the effects of LFM and LFM-A12 on invasiveness ofMDA-MB-231 cells, exponentially growing cells were incubated with eachcompound at various concentrations ranging from 12.5 RM to 100 μM in0.1% DMSO overnight. The cells were trypsinized, washed twice withserum-free DMEM containing BSA, counted and resuspended at 1×10⁵cells/ml.

[0173] A 0.5 ml cell suspension containing 5×10⁴ cells in a serum-freeDMEM containing LFM, LFM-A12, or vehicle was added to theMatrigel-coated and rehydrated filter inserts. Next, 750 μl of NIHfibroblast conditioned medium was placed as a chemoattractant in 24-wellplates and the inserts were placed in wells and incubated at 37° C. for48 hours. After the incubation period, the filter inserts were removed,the medium was decanted off and the cells on the top side of the filterthat did not migrate were scraped off with a cotton-tipped applicator.The invasive cells that migrated to the lower side of the filter werefixed, stained with Hema-3 solutions and counted under microscope. Nocells were detected at the bottom of the Boyden chambers. Therefore, thenumber of cells on the lower side of the filters accounted for all cellsthat had migrated through the filter. Five (5) to ten (10) random fieldsper filter were counted to determine the mean (±SE) values for theinvasive fraction. The invasive fractions of cells treated with LFM orLFM-A12 were compared to those of vehicle (0.1% DMSO in PBS) treatedcontrol cells and the percent inhibition of invasiveness was determinedusing the following formula: % Inhibition=100×(1-Invasive Fraction ofDrug-Treated Cells/Invasive Fraction of Control Cells).

RESULTS

[0174] Specific Inhibition of the EGFR Tyrosine Kinase by LFM-A12

[0175] The effects of LFM analogs on the enzymatic activity of the EGFRkinase in cell-free immune complex kinase assays were examined. As shownin FIG. 5A, a one hour incubation with LFM or LFM-A12 inhibited the EGFRtyrosine kinase in a dose-dependent fashion in anti-EGFRimmunoprecipitates from lysates of MDA-MB-231 human breast cancer cells.The IC₅₀ values for EGFR inhibition were 5.4 μM for LFM and 1.7 μM forLFM-A12 (FIG. 5A). In contrast, the IC₅₀ values for all other compoundswere >100 μM, except for LFM-A7 which was 74.5 μM.

[0176] The effects of LFM and LFM analogs on the enzymatic activity ofthe EGFR tyrosine kinase in breast cancer cells were also examined.After a 24 hour exposure to LFM or LFM analogs, MDA-MB-231 cells werestimulated with EGF for 10 minutes, and EGFR immune complexes from wholecell lysates were subjected to Western blot analysis with a polyclonalanti-phosphotyrosine antibody to measure the autophosphorylation of theEGFR. Treatment of MDA-MB-231 cells with LFM-A12 and, albeit to a lesserextent, with LFM resulted in decreased tyrosine phosphorylation of theEGFR after EGF stimulation. In contrast, none of the other LFM analogstested side-by-side were able to inhibit the EGFR kinase in cell-free(Table 2) or cellular ( not shown) EGFR kinase inhibition assays. Takentogether, these experimental results are consistent with the generaltrend for most of the calculated K_(i) values shown in Table 2, therebyconfirming the predictive value of the constructed homology model of theEGFR kinase domain.

[0177] In MTT assays (Uckun, F. M., et al. Clin. Can. Res. 1998, 4, 901;Narla, R. K., et al. Clin. Can. Res 1998, 1405), LFM-A12 exhibitedsignificant cytotoxicity against the MDA-MB-361 human breast cancer cellline with mean IC₅₀ value of 26.3 μM. By comparison, LFM wassignificantly less active against these breast cancer cells. The IC₅₀value for the LFM composite dose survival curves was 190.5 μM forMDA-MB-361 cells.

[0178] The inhibitory effects of LFM and LFM-A12 on the EGFR tyrosinekinase were specific in that they did not affect the enzymatic activityof other protein tyrosine kinases, including receptor family tyrosinekinase IRK (FIG. 7B), Src family tyrosine kinase HCK (FIG. 7C), Januskinases JAK1 and JAK3 (FIGS. 7D-E), and Tec family tyrosine kinase BTK(FIG. 7F), at concentrations as high as 350 μM (Table 3).

Table 3. LFM-A12 interaction scores, calculated K_(i) values andmeasured IC₅₀ values for the inhibition of several protein tyrosinekinases.

[0179] TABLE 3 LFM-A12 interaction scores, calculated K_(i) values andmeasured IC₅₀ values for the inhibition of several protein tyrosinekinases.

Lipo Ludi Ludi^(b) Inhibition Protein HB^(a) Score Score K_(i)(μM)IC₅₀(μM) EGFR 1 510 489 13 1.7 BTK 1 496 457 27 >175 IRK 1 451 41178 >350 HCK 0 510 385 142 >350 JAK1 1 387 347 340 >175 JAK3 0 402 2771710 >350

[0180] Modeling studies were performed using the crystal structurecoordinates of HCK (Sicheri et al., 1997, Nature, 385:602-9) and IRK(Hubbard, 1997, the EMBO Journal, 16:5572-5581) and constructed homologymodels for the kinase domains of JAK1, JAK3 ((Sudbeck et al., 1999,Clin. Can. Res. (in press)) and BTK (Mahajan et al., 1999, JBC, inpress) to identify possible causes for the observed selectiveity ofLFM-A12 for the EGFR tyrosine kinase. While most of the catalytic siteresidues of the EGFR kinase domain were conserved realtive to otherPTKs, we noted a few specific variatoins. EGFR residues Leu⁶⁹⁴, Val⁷⁰²,Lys⁷²¹, and Ala⁷¹⁹ are conserved in EGFR, HCK, FGFR and IRK. ResiduesAsn⁸¹⁸ and Asp⁸³¹ (opposite to hinge) are converved in EGFR, HCK, FGFR,IRK, BTK, JAK1 and JAK3. Residues Cys⁷⁵¹ and Thr⁸³⁰ are specific forEGFR but vary in BTK (Val, Ser), JAK1 (Val, Gly), JAK3 (Val, Ala), IRK(Val, Gly), and HCK (Val, Ala). Residues Thr⁷⁶⁶ and Leu⁷⁶⁸ in the hingeregion changes to Met and Leu in IRK, Met and Phe in JAK1, Met and Tyrin JAK3, and to Thr and Tyr in BTK. One region of the binding pocketcontains Cys⁷⁷³ in EGFR and is therefore considerably more hydrophobicthan the corresponding residue of PDGFR (Asp), FGFR (Asn), JAK1 (Ser),HCK (Ser), and IRK (Asp).

[0181] LFM-A12 was docked into the kinase domains of IRK, HCK, JAK3,JAK1, BTK and EGFR. After energy minimization the compound maintainedfavorable close contacts with the hinge region of each kinase althoughthe orientation of LFM-A12 in the catalytic site was different for BTKand HCK as shown in FIG. 6A. When bindign with EGFR the inhibitorappeared to be sanwiched between four residus, Leu⁶⁹⁴ and Val⁷⁰² fromabove and Leu⁸²⁰ and Thr⁸³⁰ from below. The nitrile nitrogen of theligand was involved in hydrogen bonding with the amide NH of Met⁷⁶⁹. Inaddition, the para-substituted OCF₃ group on the lead compound appearedto form close contacts between residues Thr⁷⁶⁶ and Asp⁸³¹ at positions Aand C, respectively, of EGFR (FIG. 6B).

[0182] Table 3 shows the interaction scores, estimated K_(i) values, andmeasured IC₅₀ data for LFM-A12 with the different kinases. The dataindicated that the selectivity of LFM-A12 for EGFR likely results fromits molecular shape and from favorable interactions with unique EGFRresidues that are not present in the kinase domains of the other PTKs.Likewise, unfavorable interactions with unique residues of the otherPTKs that are not found in the EGFR kinase domain also contribute tothis selectivity. These residue differences are illustrated in FIGS. 6Aand 6B at positions A and B.

[0183]FIG. 6A shows the backbone of the EGFR catalytic site, the residuedifferences between EGFR (white) and other kinases, and the dockedposition of LFM-A12 (multi-color) at this site in BTK (peach) which isalso similar to the docked position in HCK (blue). FIG. 6B shows thedocked position of LFM-A12 (multi-color) in EGFR (white), which is alsosimilar to the docked position in JAK3 (pink) and IRK (green). Thedotted surface area in each figure represents the Connolly surface ofthe inhibitor LFM-A12.

[0184] The aromatic residue in BTK (Tyr), HCK (Phe), JAK1 (Phe), JAK3(Tyr) (shown at position B in FIGS. 6A and 6B, is not as favorable forinteractions with the p-OCF₃ group of LFM-A12. The corresponding residuein the EGFR kinase domain is leucine (shown in white at position B inFIGS. 6A and 6B), which would not cause such unfavorable interactionswith LFM-A12. Also, for HCK there is a loss of hydrogen bondinginteraction with LFM-A12. Furthermore, JAK3, IRK (shown in FIG. 6B), andJAK1 (not shown) contain a methionine residue (at position A in FIG. 6B)which protrudes into the active site and could impair the closehydrophobic contact of LFM-A12 with the hinge region of the catalyticsite. The longer methionine residue in JAK3 and IRK does not complimentthe shape of LFM-A12 and may hinder its binding. As shown in FIG. 6B,the corresponding residue in the EGFR kinase domain is threonine(white); its relatively shorter side chain enables LFM-A12 (multicolor)to have a more favorable hydrophobic contact with the hinge region whichmay result in tighter binding to the EGFR binding site. For EGFR, themost active compound (LFM-A12) appears to be located between theresidues at positions A and C. Consequently, the estimated K_(i) valuefor the EGFR (13 μM) was lower than the K_(i) values for other PTKswhich ranged from 27 μM for BTK to 1710 μM for JAK3 (Table 3).

[0185] Confocal Imaging

[0186] The effects of LFM-A12 treatment on MDA-MB-231 cells wereexamined by confocal laser scanning microscopy. Slides were examinedusing a Bio-Rad MRC-1024 Laser Scanning Confocal Microscope mounted on aNikon Eclipse E800 upright microscope with high numerical apertureobjectives. Digital data was processed using Lasersharp (Bio-Rad,Hercules Calif.) and Adobe Photoshop software (Adobe Systems, MountainView, Calif.) and printed on a Pictography printer (Fuji, Elmsford,N.Y.). Bohm, H. J. J. Comput. Aided Mol. Des., 1994, 8, 243-256.

[0187] As shown in FIG. 8A, vehicle (DMSO) treated control cells wereround and large with many well organized microtubules (greenfluorescence secondary to tubulin staining) in the cytoplasm. Nuclei(blue fluorescence secondary to TOTO-3 staining) were also round andhomogenous. In contrast, MDA-MB-231 cells treated with 100 μM LFM-A12for 24 hours were much smaller and had an abnormal shape with largecytoplasmic vacuoles (FIG. 8B). The microtubules of LFM-A12 treatedcells were fewer in number and they appeared less organized than thoseof DMSO treated controls. The nuclei (blue) of the LFM-A12 treated cellswere also smaller and misshapen. Unlike LFM-A12, 100 EM LFM-A3 did notaffect the morphology or microtubular organization of MDA-MB-231 cells(FIG. 8C).

[0188] Apoptosis Assays

[0189] The morphologic features of LFM-A12 treated MDA-MB-231 cells byimmunocytohemistry (i.e., shrinkage, nuclear condensation, and abnormalmicrotubular organization) suggested that these cells might beundergoing apoptosis. Therefore, to determine whether LFM-A12 couldtrigger apoptosis in breast cancer cells, a quantitative flow cytometricapoptosis detection assay was performed.

[0190] Loose packing of membrane phospholipid head groups and cellshrinkage precede DNA fragmentation in apoptotic cells, therebyproviding MC540 binding as an early marker for apoptosis (Uckun, F. M.et al., Science, 1995, 267, 886-891). MC540 binding and propidium iodide(PI) permeability of MDA-MB-231 breast cancer cells were simultaneouslymeasured before and after a 24 hour treatment with 100 μM or 500 μMLFM-A12. Whole cells were analyzed with a FACStar Plus flow cytometer(Becton Dickinson, San Jose, Calif.). Whereas less than 5% of MDA-MB-231cells showed apoptotic changes after DMSO treatment, a significantportion of cells underwent apoptosis within 24 hours after LFM-A12treatment (Apoptotic fraction [AF] with MC540⁺/PI⁺ advanced stageapoptosis: 54% at 100 μM and 85% at 500 μM) (FIG. 9). LFM, albeit to alesser extent, also induced apoptosis in MDA-MB-231 cells.

[0191] Clonogenic Assays

[0192] The anti-cancer activity of LFM and LFM-A12 against MDA-MB-361and MDA-B-231 breast cancer cells was tested using in vitro clonogenicassays. As shown in Table 4, 24 hour treatment with LFM or LFM-A12inhibited the clonogenic growth of MDA-MB-361 cells as well asMDA-MB-231 cells in a dose-dependent fashion. At 100 μM, our leadcompound LFM-A12 killed 87.3% of clonogenic MDA-MB-361 cells and >99% ofclonogenic MDA-MB-231 cells. TABLE 4 In Vitro Anti-Tumor Activity of LFMand LFM-A12 Against Clonogenic Breast Cancer Cells Tumor CellColonies/10⁵ Cell Line Treatment Cells % Inhibition MDA-MB-361 None 1104(924, 1284) 0 DMSO (0.1%) 1088 (872, 1304) 1.4 LFM 0.1 μM 803 (702, 904)27.3 10 μM 535 (386, 684) 51.5 100 μM 196 (128, 264) 82.3 LFM-A12 0.1 μM746 (316, 1276) 32.4 10 μM 440 (276, 604) 60.2 100 μM 140 (58, 222) 87.3MDA-MB-231 None 1150 (1096, 1204) 0 DMSO (0.1%) 953 (888, 1018) 17.1 LFM0.1 μM 964 (588, 1340) 16.2 10 μM 642 (572, 712) 44.2 100 μM 297 (170,424) 74.2 LFM-A12 0.1 μM 667 (454, 880) 42.0 10 μM 515 (420, 610) 55.2100 μM 0 >99

[0193] Effects of LFM-A12 on MDA-MB-231 Breast Cancer Cell InvasionThrough Matrigel Matrix

[0194] Matrigel matrix is made up of growth factors and severalextracellular matrix (ECM) components, including collagens, laminin andproteoglycans. As shown in FIGS. 7A and 7B, MDA-MB-231 human breastcancer cells were highly invasive in Matrigel-coated Boyden chamber(CON). LFM-At2 inhibited the invasion of MDA-MB-231 cells through theMatrigel matrix in a dose-dependent fashion with an IC₅₀ value of 28.4μM and it was more potent than LFM which had IC₅₀ value of 97.0 FM(FIGS. 7A and 7B).

EXAMPLE 6 Design of New Analogs

[0195] Exploring the Catalytic Site of EGFR for the Design of SpecificInhibitors

[0196] The binding volume of the EGFR catalytic site is much larger thanthe volume occupied by the lead compound LFM-A12. Increasing the size ofthe ligand is postulated to increase the contact area between thereceptor and ligand and hence enhance binding.

[0197] Structural and chemical features of LFM analogs which aid bindingto the EGFR catalytic site are described below and illustrated in FIGS.10-11. Table 5 shows the residue differences at the ATP binding sitebetween the six PTK's : EGFR, Btk, Hck, Jak1, Jak3 and IR. TABLE 5Residue differences between EGFR, Btk, Hck, Jak1, Jak3 and IR at the ATPbinding pocket. No EGFR Btk Jak1 Jak3 IR Hck 1 Cys 751 Val Val Val ValVal 2 Leu 764 Ile Leu Leu Val Ile 3 Thr 766 Thr Met Met Met Thr 4 Leu768 Tyr Phe Tyr Leu Phe 5 Cys 773 Cys Ser Cys Asp Ser 6 Arg 817 Arg ArgArg Arg Ala 7 Thr 830 Ser Gly Ala Gly Ala

[0198] T830, C751, T766, L764, L768, C773, R817 are some selectedresidues of the EGFR ATP binding site. Some or all of these residues aredifferent from the ATP binding site of Btk, IR, Jak1, Jak3 and Hck.Amongst them, the residues Thr830 and Cys751 are specific for EGFR.These nonconserved residues can be utilized for the design of morepotent and selective inhibitors of EGFR. In particular, targeting theseresidues for interaction with specific inhibitor moieties would impactthe binding and stability of the inhibitor in the binding pocket, andenhance the specificity of the inhibitor for EGFR.

[0199] Listed in Table 6 are some groups which are proposed to aidbinding to the EGFR catalytic domain, for example by providing favorablegroups for interaction with the EGFR kinase active site. TABLE 6Substitutions on LFM analogs likely to increase the binding affinity forEGFR. No Targeting Effect Substitutions 1 Cys 751 would increase theHydrophobic groups reaching specificity for EGFR about 5.5 Å down frompara-O 2 Leu 764 would not interact with Small hydrophobic groups Btk,IR and Hck approximately within 3 Å from para-O 3 Thr 766 would notinteract with Small hydrogen bonding groups Jak1, Jak3 and IRapproximately within 3 Å from ortho and meta positions of ring (facinghinge). 4 Leu 768 5 Cys 773 would not interact with Hydrophobic groupsreaching Jak1 and IR. about 3.0 Å down from OH group of ligand 6 Arg 817would not interact with Long chain charged group Hck stretching about7.0-8.0 Å from OH or O group of ligand. 7 Thr 830 would increase theHydrophobic groups reaching specificity for EGFR approximately 3.5 Ådown from ortho and meta positions of ring (opposite hinge).

[0200] Referring to FIG. 10A, the lead compound LFM-A12 is shown boundto the EGFR- kinase domain in the predicted binding mode. A secondpossible bindign mode is shown in FIG. 10B. Our modeling studiesindicate that para substituted compounds maintain good contact with thehinge region fo the receptor and appear to be good inhibitors by ourcalculations. For the para substituted compounds, a second mode ofbinding may also be possibly where the molecule is roated 180° such thatthe aromatic ring is near residues P770, F771, G772, and the chain isnew residues L764, T766, D831 (FIG. 10B).

[0201] In order to increase the affinity and specificity of the existingligang (LFM-Al2), more ineractions with the active site residues aredesired. Substitutions at positions R₁ and R₆-R₇ of formula II areexpected to lead to increased binding affinity at the catalytic site ofEGFR. This expectation is based on the observation that the catalyticsite of the EGFR kinase domain is much larger (volumne of about 500 Å³)than the volue occupied by our most potent compounds. Increasing thesize of the ligand, preferably to fill up to about ⅔ of the volume(about 400 Å³) is predicted to increase the contact area between thereceptor and ligand and thus enhance binding.

[0202] The designed compounds of formulae III-VI are expected to providesuch increased contact with the receptor, have enhanced binding, andpotent inhibitory activity.

[0203] Novel compounds designed to fit and interact with specificcontact points of the EGFR-TK binding pocket have the followingstructural formulae (III-VI):

[0204] where R₅ is H, NH₂, CH₃, OH, CF₃, or halo. Preferably, R₅ is notH, and halo is Br or Cl.

[0205] where R₅ is H, NH₂, CH₃, OH, CF₃, or halo. Preferably, R₅ is notH, and halo is F or Cl.

[0206] where R₄ is NH—CH₃ or OCH₃.

[0207] where R₁ is —CH₂—CH₂X and X is halo, preferably Cl or Br; or R₁^(_(—CH)) ₂CF₃; or R₁ is:

[0208] These compounds are synthesized as detailed in the followingschemes, according to their type of modifications. By using the propersubstituted-aniline, the compounds shown as formulae III, IV, and V maybe synthesized by the same general synthetic pathway used above forExample 4 (Scheme 1). Synthetically, the entry for synthesizing thecompounds of formula VI is to use different acylating agent, acidchloride, in the last step of the synthetic pathway shown in Scheme 1.The following schemes illustrate the four general types ofmodifications, and general synthesis schemes for the desiredsubstituted-analines for the four types.

[0209] All publications, patents, and patent documents are incorporatedby reference herein, as though individually incorporated by reference.The invention has been described with reference to various specific andpreferred embodiments and techniques. However, it should be understoodthat many variations and modifications may be made while remainingwithin the spirit and scope of the invention.

We claim:
 1. A method for inhibiting EGFR tyrosine kinase, the methodcomprising contacting the EGFR with a compound having the structuralformula:


2. A method for inhibiting EGFR tyrosine kinase without inhibiting Srcfamily tyrosine kinases, Tec family tyrosine kinases, or Janus familytyrosine kinases, the method comprising contacting the EGFR with acompound having the structural formula:


3. A method of treating cancer cells that express EGFR, the methodcomprising contacting the cancer cells that express EGPR with a compoundhaving the structural formula: